The proteasome and 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. Under the control of interferon-gamma the proteolytic properties of the proteasome are adapted to the requirements of the immune system. Interferon-gamma induces the formation of immunoproteasomes and the synthesis of the proteasome activator PA28. Both alter the proteolytic properties of the proteasome complex and enhance proteasomal function in antigen presentation. Thus, a combination of several of regulatory events tunes the proteasome system for maximal efficiency in the generation of MHC class I antigens.     

Modulators of the activation of the proteasome by PA28 (11S Reg)

The degradation of chromogenic substrates and oligopeptides by the 20S proteasome is markedly enhanced and the generation of antigens for presentation by the MHC class-I system is facilitated by combination with an activator protein known as PA28 or 11S reg. We have described the properties of a PA28-proteasome modulator, N-benzyloxycarbonyl-Ile-Glu(O-t-Bu)-Ala-leucinol which shifts the pathway of peptide hydrolysis by the activated proteasome to products terminating in an acidic amino acid at the expense of products terminating in a hydrophobic amino acid. We now report that piperazinyl phenothiazines and several other antipsychotic drugs modulate the PA28-20S activated proteasome in an opposite manner. Fluphenazine, trifluoperazine and prochlorperazine antagonize the peptidylglutamyl peptide bond hydrolyzing activity of the activated proteasome much more strongly than the chymotrypsinlike activity. The chicken ovalbumin immunodominant epitope SIINFEKL is degraded by the activated proteasome to SIINFE and SIINF in approximately equimolar amounts. Piperazinyl phenothiazines promote formation of SIINF whereas Psi-ol promotes formation of SIINFE. PA28- proteasome modulators by modifying the profile of peptides produced by the activated proteasome, may either enhance or suppress the immune response.     

Hsp90-mediated assembly of the 26 S proteasome is involved in major histocompatibility complex class I antigen processing

Heat shock protein 90 (hsp90) and the proteasome activator PA28 stimulate major histocompatibility complex (MHC) class I antigen processing. It is unknown whether hsp90 influences the proteasome activity to produce T cell epitopes, although association of PA28 with the 20 S proteasome stimulates the enzyme activity. Here, we show that hsp90 is essential in assembly of the 26 S proteasome and as a result, is involved in epitope production. Addition of recombinant hsp90alpha to cell lysate enhanced chymotrypsin-like activity of the 26 S proteasome in an ATP-dependent manner as determined by an in-gel hydrolysis assay. We successfully pulled down histidine-tagged hsp90alpha- and PA28alpha-induced, newly assembled 26 S proteasomes from the cell extracts for in vitro epitope production assay, and we found these structures to be sensitive to geldanamycin, an hsp90 inhibitor. We found a cleaved epitope unique to the proteasome pulled down by both hsp90alpha and PA28alpha, whereas two different epitopes were identified in the hsp90alpha- and PA28alpha-pulldowns, respectively. Processing of these respective peptides in vivo was enhanced faithfully by the protein combinations used for the proteasome pulldowns. Inhibition of hsp90 in vivo by geldanamycin partly disrupted the 26 S proteasome structure, consistent with down-regulated MHC class I expression. Our results indicate that hsp90 facilitates MHC class I antigen processing through epitope production in a complex of the 26 S proteasome.     

Regulation of proteasome complexes by gamma-interferon and phosphorylation

Proteasomes play a major role in non-lysosomal proteolysis and also in the processing of proteins for presentation by the MHC class I pathway. In animal cells they exist in several distinct molecular forms which contribute to the different functions. 26S proteasomes contain the core 20S proteasome together with two 19S regulatory complexes. Alternatively, PA28 complexes can bind to the ends of the 20S proteasome to form PA28-proteasome complexes and PA28-proteasome-19S hybrid complexes have also been described. Immunoproteasome subunits occur in 26S proteasomes as well as in PA28-proteasome complexes. We have found differences in the subcellular distribution of the different forms of proteasomes. The gamma-interferon inducible PA28 alpha and beta subunits are predominantly located in the cytoplasm, while 19S regulatory complexes (present at significant levels only in 26S complexes) are present in the nucleus as well as in the cytoplasm. Immunoproteasomes are greatly enriched at the endoplasmic reticulum (ER) where they may facilitate the generation of peptides for transport into the lumen of the ER. We have also investigated the effects of gamma-interferon on the levels and subcellular distribution of inducible subunits and regulator subunits. In each case gamma-interferon was found to increase the level but not to alter the distribution. Several subunits of proteasomes are phosphorylated including alpha subunits C8 (alpha7) and C9 (alpha3), and ATPase subunit S4 (rpt2). Our studies have shown that gamma-interferon treatment decreases the level of phosphorylation of proteasomes. We have investigated the role of phosphorylation of C8 by casein kinase II by site directed mutagenesis. The results demonstrate that phosphorylation at either one of the two sites is essential for the association of 19S regulatory complexes and that the ability to undergo phosphorylation at both sites gives the most efficient incorporation of C8 into the 26S proteasome.     

The N-terminal flanking region of the TRP2360-368 melanoma antigen determines proteasome activator PA28 requirement for epitope liberation

Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens. The interferon-gamma-inducible proteasome activator PA28 plays an important role in the generation of MHC ligands by proteasomes. Generation of the HLA-A(*)0201 restricted melanoma antigen TRP2(360-368) by the proteasome has been shown to be dependent on the function of PA28 in vitro and in vivo (Sun, Y., Sijts, A. J., Song, M., Janek, K., Nussbaum, A. K., Kral, S., Schirle, M., Stevanovic, S., Paschen, A., Schild, H., Kloetzel, P. M., and Schadendorf, D. (2002) Cancer Res. 62, 2875-2882). Here we analyzed the role of the epitope sequence environment in determining this PA28 dependence. Experiments using the melanoma TRP2(288-296) epitope and the murine cytomegalovirus-derived pp89 epitope precursor peptide for epitope replacement revealed that the TRP2(360-368) flanking sequences can transfer PA28 dependence onto otherwise PA28 independent epitopes. Moreover, the N-terminal flanking sequence is sufficient to establish PA28 dependence of an epitope by allowing PA28-induced coordinated dual cleavages. These results show that N-terminal flanking sequences strongly influence epitope generation efficiency and that PA28 function is particularly relevant for the generation of normally poorly excised peptide products.     

26 S proteasome-mediated production of an authentic major histocompatibility class I-restricted epitope from an intact protein substrate.

Peptides displayed on the cell surface by major histocompatibility class I molecules (MHC class I) are generated by proteolytic processing of protein-antigens in the cytoplasm. Initially, antigens are degraded by the 26 S proteasome, most probably following ubiquitination. However, it is unclear whether this proteolysis results in the generation of MHC class I ligands or if further processing is required. To investigate the role of the 26 S proteasome in antigen presentation, we analyzed the processing of an intact antigen by purified 26 S proteasome. A recombinant ornithine decarboxylase was produced harboring the H-2K(b)-restricted peptide epitope, derived from ovalbumin SIINFEKL (termed ODC-ova). Utilizing recombinant antizyme to target the antigen to the 26 S proteasome, we found that proteolysis of ODC-ova by the 26 S proteasome resulted in the generation of the K(b)-ligand. Mass spectrometry analysis indicated that in addition to SIINFEKL, the N-terminally extended ligand, HSIINFEKL, was also generated. Production of SIINFEKL was linear with time and directly proportional to the rate of ODC-ova degradation. The overall yield of SIINFEKL was approximately 5% of the amount of ODC-ova degraded. The addition of PA28, the 20 S, or the 20 S-PA28 complex to the 26 S proteasome did not significantly affect the yield of the antigenic peptide. These findings demonstrate that the 26 S proteasome can efficiently digest an intact physiological substrate and generate an authentic MHC class I-restricted epitope.     

Pattern of MHC class I and immune proteasome expression in Walker 256 tumor during growth and regression in Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis

Dynamics of the expression of MHC class I, immune proteasomes and proteasome regulators 19S, PA28, total proteasome pool and proteasome chymotrypsin-like activity in Walker 256 tumor after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis was studied. The tumor growth and regression in Brattleboro rats were accompanied by changes in the proteasome subunit level unlike the tumor growth in WAG rats with normal expression of arginine-vasopressin gene. In the tumor implanted into Brattleboro rats the immune proteasome level was maximal between days 14 and 17, when the tumor underwent regression. Conversely, the expression of proteasome regulators tended to decrease during this period. Immune proteasomes are known to produce antigen epitopes for MHC class I to be presented to CD8+ T lymphocytes. Enhanced expression of immune proteasomes coincided with the recovery of MHC class I expression, suggesting the efficient presentation of tumor antigens in Brattleboro rats.     

Trypanosoma cruzi: effect of the infection on the 20S proteasome in non-immune cells

Human infection with the protozoan Trypanosoma cruzi leads to Chagas disease. After 10-20 years of the normal acute phase, this disease develops to a chronic phase characterized mainly by dilated congestive cardiomyopathy. The mechanisms involved in the chronic phase are poorly understood, and it has been suggested that the parasite evades immune surveillance by down regulating the MHC class I antigen processing pathway. Here we analyzed whether composition or expression of the 20S proteasome, the major proteinase responsible for the generation of MHC class I ligands, were altered upon infection of HeLa cells by T. cruzi. Two-dimensional gel electrophoresis and RT-PCR experiments comparing non-infected and infected cells did not show differences between the composition of 20S proteasome or expression of its subunits. However, the proteasome’s trypsin- and chymotrypsin-like activities were 2.5 and 3.6 times higher in infected cells than in non-infected cells. Our results suggest that in vitroT. cruzi infection of human or rat cells do not alter the expression of 20S proteasomal subunits or particle composition, and fails to induce the formation of immunoproteasome. However, a significant increase in the trypsin- and chymotrypsin-like activities of the host proteasome was observed.     

Hepatitis B virus HBx peptide 116-138 and proteasome activator PA28 compete for binding to the proteasome alpha4/MC6 subunit

PA28 is a modulator of the 20S proteasome. The PA28 binding sites on the 20S proteasome are still not well defined. Using yeast two-hybrid interaction assays and proteasome inactivation kinetics we provide evidence that the proteasome alpha4 subunit is one of the PA28 binding sites. This finding is supported by the observation that a hepatitis B virus X protein-derived polypeptide habouring the alpha4 proteasome subunit binding motif impairs the activation of 20S proteasomes by PA28.     

PA28 subunits of the mouse proteasome: primary structures and chromosomal localization of the genes

 The 20S proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex (MHC) class I molecules. Recent evidence indicates that an interferon-γ (IFN-γ)-inducible PA28 activator complex enhances the generation of class I binding peptides by altering the cleavage pattern of the proteasome. In the present study, we determined the primary structures of the mouse PA28 α- and β-subunits. The deduced amino acid sequences of the α- and β-subunits were 49% identical. We also determined the primary structure of the mouse PA28 γ-subunit (Ki antigen), a protein of unknown function structurally related to the α- and β-subunits. The amino acid sequence identity of the γ-subunit to the α- and β-subunits was 40% and 32%, respectively. Interspecific backcross mapping showed that the mouse genes coding for the α- and β-subunits (designated Psme1 and Psme2, respectively) are tightly linked and map close to the Atp5g1 locus on chromosome 14. Thus, unlike the LMP2 and LMP7 subunits, the IFN-γ-inducible subunits of PA28 are encoded outside the MHC. The gene coding for the γ-subunit (designated Psme3) was mapped to the vicinity of the Brca1 locus on chromosome 11. A computer search of the DNA databases identified a γ-subunit-like protein in ticks and Caenorhabditis elegans, the organisms with no adaptive immune system. It appears that the IFN-γ-inducible α- and β-subunits emerged by gene duplication from a γ-subunit-like precursor. Received: 11 March 1997     

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.     

Inactivation of the 20S proteasome in Mycobacterium smegmatis

The 20S proteasome is an essential component of the cytosolic protein turnover apparatus of eukaryotic cells. In higher eukaryotes, the 20S proteasome is responsible for most cytosolic protein turnover and also generates peptides for subsequent presentation by the MHC class I pathway. Structurally, the eukaryotic 20S proteasome is extremely complex, being composed of 14 different subunits. Proteasomes with simplified subunit composition have been identified in certain eubacteria and archaebacteria but, in each case, the proteasome-containing organism is recalcitrant to further molecular genetic analyses. As a result, no in vivo characterization of a simplified eubacterial or archaebacterial proteasome has been reported. We have shown that the genetically tractable eubacterium Mycobacterium smegmatis contains a 20S proteasome, allowing the first in vivo characterization of a simplified 20S proteasome. We use a positive/negative selection scheme to inactivate the genes encoding 20S proteasome subunits and demonstrate that, in contrast to eukaryotic cells, M. smegmatis cells lacking intact proteasome genes are viable and phenotypically indistinguishable from congenic strains containing proteasomes. Implications for the evolution of the protein turnover apparatus are discussed.     

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-γ. 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-γ 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.     

Characterization of the 20S proteasome of the lepidopteran,Spodoptera frugiperda

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.     

Changes in proteasome expression and activity during differentiation of neuronal precursor NTera 2 clone D1 cells

The effect of differentiation of the human neuronal progenitor cell line NTera 2 clone D1 (NT2/D1) by retinoic acid on components of the proteasome system was studied. The chymotrypsin-like and peptidylglutamyl peptide bond hydrolyzing activities of the proteasome increased five weeks after retinoic acid, and following treatment with mitotic inhibitors returned to levels detected in non-differentiated cells. A selective induction of the MHC class II region encoded LMP7 and LMP2 proteasome subunits occurred during differentiation, whereas there were no changes in the expression of the constitutive LMP2 counterpart (delta-subunit) or the constitutive C2 subunit. Immunofluorescence revealed marked LMP7 accumulation in fully differentiated cells, with no changes in the labeling pattern of the constitutive proteasome antigens. The expression of the alpha-subunit of the PA28 proteasome activator was down-regulated in fully differentiated neurons, but was not correlated with changes in enzymatic activity. Changes in proteasome activity and composition may contribute to the processes leading to differentiation of human neurons in vitro and to the properties of fully differentiated neurons.     

Proteasomes in the brain of β2-microglobulin knockout mice

MHC class I molecules play an important role in synaptic plasticity of the mammalian nervous system. Proteolytic complexes (proteasomes) produce oligopeptides that are presented on cell surfaces in complexes with MHC class I molecules and regulate many cellular processes beside this. The goal of the present work was to study peculiarities in functioning of proteasomes and associated signaling pathways along with evaluation of NeuN and gFAP expression in different sections of the brain in mice with knockout of β2-microglobulin, a constituent of MHC class I molecules. It was found that the frontal cortex and the brainstem, structures with different ratio of NeuN and gFAP expression, are characterized by opposite changes in the proteasome pool under constant total proteasome levels in B2m-knockout mice in comparison with those in control animals. ChTL-activity as well as expression of LMP7 immune subunit and PA28 regulator of proteasomes was elevated in the cortex of B2m-knockout mice, while these indicators were decreased in the brainstem. The concentrations of the signaling molecules nNOS and HSP70 in B2m-knockout mice were increased in the cortex, while being decreased in the brainstem, and this indicates the possibility of control of expression of the LMP7 subunit and the regulator PA28 by these molecules. Changes in the proteasome pool observed in striatum of B2m-knockout mice are similar to those observed in the brainstem. At the same time, the cerebellum is characterized by a specific pattern of proteasome functioning in comparison with that in all other brain structures. In cerebellum the expression of immune subunits LMP7 and LMP2 and the regulator PA28 was increased, while expression of regulator PA700 was decreased. Deficiency of NeuN and gFAP was revealed in most brain compartments of B2m-knockout mice. Thus, increased expression of the above-mentioned immune subunits and the proteasome regulator PA28 in the cortex and cerebellum may compensate disturbances revealed in the brain structures and the absence of MHC class I molecules. Apparently, this promotes production of peptides necessary for cell-to-cell interactions and maintains nervous system plasticity in B2m-knockout mice.     

Nitric oxide decreases activity and levels of the 11S proteasome activator PA28 in the vasculature

The 11S proteasome activator (PA28) binds to the 20S proteasome and increases its ability to degrade small peptides. Expression of PA28 subunits (α, β, γ) is induced by interferon-γ stimulation. Inflammation plays a role in the development of neointimal hyperplasia, and we have previously shown that nitric oxide (NO) reduces neointimal hyperplasia in animal models and 26S proteasome activity in rat aortic smooth muscle cells (RASMC). Here, we show that PA28 increased 26S proteasome activity in RASMC, as measured by a fluorogenic assay, and the NO donor S-nitroso N-acetylpenicillamine significantly inhibits this activation. This effect was abrogated by the reducing agents dithiothreitol and HgCl(2), suggesting that NO affects the activity of PA28 through S-nitrosylation. NO did not appear to affect PA28 levels or intracellular localization in RASMC in vitro. Three days following rat carotid artery balloon injury, levels of PA28α, β and γ subunits were decreased compared to uninjured control arteries (n=3/group) in vivo. The NO donor proline NONOate further decreased PA28α, β and γ levels by 1.9-, 2.3- and 3.4-fold, respectively, compared to uninjured control arteries. Fourteen days following arterial injury, levels of PA28α, β and γ subunits were increased throughout the arterial wall compared to uninjured control arteries, but were greatest for the α and β subunits. NO continued to decrease the levels of all three PA28 subunits throughout the arterial wall at this time point. Since the PA28 subunits are involved in the breakdown of peptides during inflammation, PA28 inhibition may be one mechanism by which NO inhibits neointimal hyperplasia.     

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.