Control of LMP7 expression in human endothelial cells by cytokines regulating cellular and humoral immunity

Formation of antigenic peptides by the multicatalytic proteinase complex (MPC, proteasome) is facilitated by incorporation of three subunits (LMP2, LMP7 and LMP10) that are inducible by IFN-gamma and TNF-alpha. These cytokines, or their functional homologues (e.g. TNF-beta), are released from many cells including Th(1)lymphocytes. To learn more about the relationship between control of cellular immunity and expression of LMP subunits, we measured LMP7 levels in human umbilical vein endothelial cells of cytokines promoting cellular immunity (IL-12, IFN-gamma, TNF-alpha) or humoral immunity (IL-10, IL-6). Little or no effect was seen when cells were exposed to IL-6, IL-10 or IL-12 alone. IFN-gamma upregulated LMP7 levels, as did TNF-alpha to a lesser extent. IL-10 downregulated IFN-gamma-induced increases in LMP7 levels, as did IL-12. The findings indicate that regulation of levels of LMP7 is similar to and may be coupled with that of other molecules required for MHC class I-dependent immunity, and depends primarily on cytokines released by Th(1)helper lymphocytes.     

Components of the bovine pituitary multicatalytic proteinase complex (proteasome) cleaving bonds after hydrophobic residues.

Two catalytic components of the multicatalytic proteinase complex (MPC, proteasome) designated as chymotrypsin-like (ChT-L) and branched chain amino acid preferring (BrAAP) cleave bonds after hydrophobic amino acids. The possible involvement of the ChT-L and peptidylglutamyl-peptide hydrolyzing (PGPH) activities in the cleavage of bonds attributed to the BrAAP component was examined. Several inhibitors of the ChT-L activity containing a phenylalaninal group did not affect the BrAAP activity at concentrations that were more than 150 times higher than their K(i) values for the ChT-L activity. Concentrations of lactacystin that inactivated more than 90% of the ChT-L activity had no effect on the BrAAP activity. Concentrations of 3,4-dichloroisocoumarin (DCI) that inactivated the ChT-L activity activated by up to 10-fold the BrAAP activity toward synthetic substrates and by more than 2-fold the degradation of the insulin B chain in a reaction not inhibited by Z-LGF-CHO, a selective inhibitor of the ChT-L activity. These findings are incompatible with any significant involvement of the ChT-L activity in the cleavage of BrAAP substrates. Both the native and DCI-treated MPC cleaved the insulin B chain mainly after acidic residues in a reaction inhibited by Z-GPFL-CHO, an inhibitor of the BrAAP and PGPH activities. DCI exposure did not result in acylation of the N-terminal threonine in the active site of the Y subunit. These results suggest involvement of the PGPH activity in the cleavage of BrAAP substrates, but this conclusion is incompatible with DCI activation of the BrAAP activity and inactivation of the PGPH activity, and with the finding that proteins inhibiting the PGPH activity had no effect on the BrAAP activity. Rationalization of these contradictions is discussed.     

Peroxynitrite-induced oxidation and its effects on isolated proteasomal systems

The proteasomes are the major intracellular proteolytic systems involved in the removal of altered proteins. In this study, we examined different susceptibilities of constitutive (XYZ) and interferon-gamma inducible (LMP) 20S proteasomes, isolated from bovine brain and thymus, respectively, to peroxynitrite-mediated oxidation. Exposure of XYZ and LMP proteasomes to increasing amounts of peroxynitrite resulted in different levels, in the two enzymes, of 3-nitrotyrosine groups and tryptophan residues oxidation. 1-Anilino-8-naphtalene-sulfonic acid binding studies and quenching of tryptophan residues indicated that the LMP complex was more sensitive to peroxynitrite. Regarding the proteolytic activities, the XYZ proteasome showed an overall activation (even if the trypsin-like (T-L) component was 20% inhibited), with the peptidyl-glutamyl peptide-hydrolyzing (PGPH) and branched-chain amino acid-preferring (BrAAP) activities being the most stimulated. On the other end, the LMP proteasome was inhibited, especially the BrAAP activity, whereas the T-L activity was not affected. Furthermore, exposure to increasing amounts of peroxynitrite induced a gradual decrease of beta-casein degrading rate by the LMP proteasome, whereas it did not influence the constitutive complex. Our results indicated that peroxynitrite caused a mild modification of the XYZ complex, leading to activation of its catalytic activities. Differently, the LMP proteasome showed a more significant conformational change resulting in the inhibition of the proteolytic functions.     

RNA degrading activity is tightly associated with the multicatalytic proteinase, ingensin

The multicatalytic proteinase, ingensin, was purified to homogeneity from chicken liver. rRNA-degrading activity was co-eluted with the purified multicatalytic proteinase from a TSK-3000SW column. This RNA-degrading activity was inactivated by heat treatment and the addition of a low concentration of SDS. Therefore, the RNA-degrading activity co-eluted with the multicatalytic proteinase was not due to contamination by low-molecular-mass RNases. These results strongly suggest that this RNA-degrading activity was tightly associated with the multicatalytic proteinase, ingensin.     

Evidence indicating that the multicatalytic proteinase of rabbit reticulocytes is not incorporated as a core enzyme into a 26 S proteinase complex.

We have reinvestigated the recent proposal that the multicatalytic proteinase, together with other components of reticulocyte lysate, may become incorporated into a very large, "26 S" proteinase complex via an ATP-dependent process. Different from these published results, we consistently isolate the multicatalytic proteinase as a 650,000 Da "20 S" multisubunit proteinase. Analysis on nondenaturing polyacrylamide gels of reticulocyte fractions containing the putative complexed form of the multicatalytic proteinase reveal that activity against succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin is associated with two groups of protein of different molecular mass. One migrates like multicatalytic proteinase purified to homogeneity, displays, on sodium dodecyl sulfate gels, a set of protein species in the range of 23,000-32,000 Da, characteristic of the multicatalytic proteinase, and is recognized by a monospecific antibody to the enzyme. In contrast, the activity associated with the higher molecular mass (26 S) proteinase complex lacks the typical multicatalytic proteinase subunits and is devoid of antigenic material, when tested with the antibody. These results confirm and extend our recent findings in mouse liver by showing that the multicatalytic proteinase is not a constituent of a 26 S proteinase complex.     

Effect of neurotoxic metal ions on the proteolytic activities of the 20S proteasome from bovine brain

The effect of oxidative stress induced by neurotoxic metal ions on the properties of the brain 20S proteasome or multicatalytic proteinase complex (MPC) has been studied. Exposure of the 20S proteasome to increasing amounts of Fe(III), Fe(II), Cu(II) or Zn(II) affects its main hydrolytic activities: trypsin-like (T-L), chymotrypsin-like (ChT-L), peptidylglutamyl-peptide hydrolase (PGPH), branched-chain amino acid preferring (BrAAP) and caseinolytic activities, although in different ways. T-L activity showed gradual activation by both iron ions but inhibition by Cu(II) and Zn(II). ChT-L and PGPH activities were inhibited whereas BrAAP activity was widely activated by all the tested metal salts except for zinc ions. Moreover, the exposure to ferrous salt increased the degradation rate of casein. The functional effects appear to be linked to oxidation-induced modifications, as demonstrated by an increase of carbonyl groups following the exposure to metal ions. In addition, modifications induced by ferrous salt on the catalytic subunits were also supported by western blot analyses performed using anti-X, anti-Y and anti-Z antibodies. The results obtained clearly indicate that metal-catalyzed oxidation strongly affects the functions of the brain 20S proteasome, even though the catalytic subunits seem to be differently influenced by oxidative phenomena.     

Interaction of human erythrocyte multicatalytic proteinase with polycations

The multicatalytic proteinase from human erythrocytes (macropain, proteasome) is a large enzyme composed of at least six distinct subunits ranging in molecular masses from 20 to 30 kDa. As its name implies, this proteinase appears to contain multiple catalytic sites with differing specificities toward peptide substrates. Several polycationic substances, including polylysines, polyarginine, protamine and histone H1 markedly stimulated caseinolytic activity of the proteinase. Activation was instantaneous, and involved increasing the Vmax of the proteinase for casein. Prolonged preincubation with polylysine at 37 degrees C resulted in autolytic inactivation of the proteinase. The polylysine concentrations required for half-maximal activation or autolytic inactivation were the same. A 23 kDa subunit of the proteinase disappeared at the same rate as loss of catalytic activity, and with the same pH dependence and polylysine concentration dependence. These results suggest that polylysine perturbs the structure of the multicatalytic proteinase, resulting in increased catalytic activity toward substrates; and, with prolonged exposure, allowing autoproteolytic inactivation to occur. The 23 kDa subunit appeared to be required for expression of caseinolytic activity, and may therefore be a catalytic subunit of the complex having activity against casein.     

26S multicatalytic proteinase complexes decrease during the differentiation of murine erythroleukemia cells.

Changes in multicatalytic proteinase activity during differentiation were investigated using Me2SO-induced differentiation of murine erythroleukemia cells as a model. The apparent ATP-dependent multicatalytic proteinase activity decreased in the Me2SO-treated cells with ATP-dependent incorporation of [3H]diisopropyl fluorophosphate decreasing notably after Me2SO-treatment. This decrease in activity does not seem to arise from a cessation of cell-proliferation, because no significant changes in proteinase activity were observed under different culture conditions. Hydroxyapatite column chromatography was employed to analyze the form of multicatalytic proteinase. It was clearly demonstrated that the 26S form of the proteinase decrease in the differentiated cells relative to normal cells. Multicatalytic proteinase-associated proteins that bind to the proteinase in an ATP-dependent manner were purified on an anti-multicatalytic proteinase IgG conjugated column. Only a small amount of protein was recovered from the differentiated cells. These results suggest that the decrease in multicatalytic proteinase-associated proteins that occurs upon cell-differentiation abolishes the ATP-dependent activity of the proteinase.     

The antiviral immune response in mice devoid of immunoproteasome activity

The replacement of the catalytically active proteasome subunits β1, β2, and β5 by the immunoproteasome subunits low molecular mass polypeptide (LMP) 2 (β1i), multicatalytic endopeptidase complex-like-1 (MECL-1) (β2i), and LMP7 (β5i) is required for the production of numerous class I ligands. Hitherto, investigation of the immunoproteasome was confined to the analysis of mice deficient for one or two immunosubunits. In this study, we characterized LMP2(-/-)/MECL-1(-/-) double-deficient mice and used the well-defined LMP7-selective inhibitor ONX 0914 in these mice to generate mice lacking the activity of all immunoproteasome subunits. LMP2(-/-)/MECL-1(-/-) double-deficient mice had strongly reduced numbers of CD8(+) T cells in the spleen. Nevertheless, infection with the lymphocytic choriomeningits virus induced a normal cytotoxic T cell response in these mice, although the T cell response to several class I epitopes was altered. Treatment of LMP2(-/-)/MECL-1(-/-) double-deficient mice with the LMP7-selective inhibitor ONX 0914 elicited a strong CTL response in lymphocytic choriomeningitis virus-infected mice. Thereby, the T(CD8+) response to nucleoprotein 205-212, which is barely detectable in LMP2(-/-)/MECL-1(-/-) double-deficient mice, could be reverted to normal levels by LMP7 inhibition. Additional experiments could demonstrate that the increased CTL response to the nucleoprotein 205-212 in mice lacking functional immunoproteasome is due to an altered class I presentation of this epitope. Taken together, to our knowledge, this is the first study investigating viral infection in mice lacking activity of all three immunoproteasome subunits.     

Coordinate increase in activity of proteinase subunits of the 1300-kDa megaproteinase of Frankia strain BR: role of carbon source depletion and extracellular metabolites

We have recently described the presence of a high molecular mass multicatalytic proteinase complex (megaproteinase; 28 S, 1300 kDa) in Frankia strain BR. The complex dissociates into 11 low molecular mass proteinase subunits (40-19 kDa) when subjected to sodium dodecyl sulfate - gelatin - polyacrylamide gel electrophoresis. We show here that the activity of these proteinase subunits strongly increased after cessation of growth in stirred BAP-PCM mineral medium. Subsequent addition of either BAP medium components or sodium propionate alone, as carbon source, to a Frankia culture at the end of the exponential growth phase was found to prolong growth for 1 additional day, and to delay the increase in activity of the proteinase subunits for 3 days after cessation of growth. Addition of ammonium chloride alone, as nitrogen source, had no effect. On the other hand, when Frankia cells in the late exponential phase (3 days) were resuspended in a culture filtrate recovered from a 5-day-old culture and supplemented with BAP-PCM medium components, the biomass yield decreased to about 50%. Also, the activity of the proteinase subunits increased as soon as growth ceased. The ability of this culture filtrate to inhibit growth and stimulate the activity of proteinase subunits was partially lost by heating or was largely removed by DEAE-cellulose treatment. Thus, our findings indicate an extracellular control of Frankia megaproteinase activity, suggesting that carbon source depletion and probably accumulation of heat-sensitive growth-inhibiting metabolites in the medium are determining factors.     

Isolation and characterization of bovine thymus multicatalytic proteinase complex

The multicatalytic proteinase complex (MPC or proteasome) from bovine thymus was isolated and purified to homogeneity applying a protocol utilizing ion exchange and gel permeation chromatography as major purification tools. The purified complex shows molecular properties that are common for proteasomal molecules (high molecular mass, multisubunit organization, and multiple proteolytic activities) even though a peculiar subunit composition and the presence of specific regulatory mechanisms affecting the assembled proteolytic activities suggest a specialized function for this complex. Thymus proteasome is characterized by the presence of LMP2, LMP7, and LMP10 (MECL1) subunits, which replace the X, Y, and Z subunits. Since a similar complex was previously isolated in bovine spleen, it appears that the proteasomal population containing the LMP subunits is characteristic for organs involved in immune response. Both the thymus and spleen proteasomes are characterized by a marked efficiency in cleaving peptide bonds after branched-chain and aromatic amino acids, indicating that this proteasomal population is most likely involved in intracellular processing of class I antigenic peptides and is an example of an "in vivo" functioning immunoproteasome. However, in spite of several similarities, the complexes isolated from the two lymphoid organs do not show superimposable functional properties, which suggests the presence of organ-specific regulatory mechanisms affecting each of the proteolytic components assembled in the complex.     

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.     

Tissue-specific changes of multicatalytic proteinase activity in the fasted rat.

During a three-day fast, followed by four days of refeeding, the content of the multicatalytic proteinase as well as hydrolyzing activity towards Suc-Leu-Leu-Val-Tyr-7-amino-4-methylocoumarin (SLLVT-MCA) was measured in various rat tissues. When compared with normal rats, the MCP content, as determined by immunochemical techniques, was unchanged over the entire experimental period in the three tissues examined: gastrocnemius muscle, thymus and testis. By contrast, a differential response was observed in the three tissues with respect to specific and total SLLVT-MCA splitting activity: for thymus and testis, these values were again unchanged, whereas in gastrocnemius muscle, both specific and total enzyme activity fell by almost 70% on day three of fasting but returned to control values on day four of refeeding. This change in activity was not due to the accumulation or degradation of a specific proteinase inhibitor. Data demonstrate that, in association with the insulin-deficient state of starvation, the activity of the multicatalytic proteinase shows an adaptive behaviour which becomes manifest in some but not in other tissues.     

Increased local vascular endothelial growth factor expression associated with antitumor activity of proteasome inhibitor

Inhibition of the proteasome, a multicatalytic proteinase complex, is an attractive approach to cancer therapy. Here we report that a selective inhibitor of the chymotrypsin-like activity of the proteasome, PSI (N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal) may inhibit growth of solid tumors not only through apoptosis induction, but also indirectly--through inhibition of angiogenesis. Two murine tumors: colon adenocarcinoma (C-26) and Lewis lung carcinoma (3LL) were chosen to study the antitumor effect of PSI. In an in vivo model of local tumor growth, PSI exerted significant antitumor effects against C-26 colon carcinoma, but not against 3LL lung carcinoma. Retardation of tumor growth was observed in mice treated with both 10 nmoles and 100 nmoles doses of PSI and in the latter group prolongation of the survival time of tumor-bearing mice was observed. PSI inhibited angiogenesis in the C-26 growing tumors with no such effect in 3LL tumors. Unexpectedly, that activity was associated with upregulation of vascular endothelial growth factor (VEGF) at the level of mRNA expression and protein production in C-26 tumors treated with PSI. C-26 cells treated with PSI produced increased amounts of VEGF in vitro in a dose- and time-dependent manner. We demonstrated that in C-26 colon adenocarcionoma higher VEGF production may render endothelial cells susceptible to the proapoptotic activity of PSI and is associated with inhibition of tumor growth.     

Why the structure but not the activity of the immunoproteasome subunit low molecular mass polypeptide 2 rescues antigen presentation

The proteasome is responsible for the generation of most epitopes presented on MHC class I molecules. Treatment of cells with IFN-γ leads to the replacement of the constitutive catalytic subunits β1, β2, and β5 by the inducible subunits low molecular mass polypeptide (LMP) 2 (β1i), multicatalytic endopeptidase complex-like-1 (β2i), and LMP7 (β5i), respectively. The incorporation of these subunits is required for the production of numerous MHC class I-restricted T cell epitopes. The structural features rather than the proteolytic activity of an immunoproteasome subunit are needed for the generation of some epitopes, but the underlying mechanisms have remained elusive. Experiments with LMP2-deficient splenocytes revealed that the generation of the male HY-derived CTL-epitope UTY(246-254) was dependent on LMP2. Treatment of male splenocytes with an LMP2-selective inhibitor did not reduce UTY(246-254) presentation, whereas silencing of β1 activity increased presentation of UTY(246-254). In vitro degradation experiments showed that the caspase-like activity of β1 was responsible for the destruction of this CTL epitope, whereas it was preserved when LMP2 replaced β1. Moreover, inhibition of the β5 subunit rescued the presentation of the influenza matrix 58-66 epitope, thus suggesting that a similar mechanism can apply to the exchange of β5 by LMP7. Taken together, our data provide a rationale why the structural property of an immunoproteasome subunit rather than its activity is required for the generation of a CTL epitope.     

Itchy,a Nedd4 ubiquitin ligase,downregulates latent membrane protein 2A activity in B-cell signaling

Nedd4 family ubiquitin protein ligases (E3s) specifically associate with latent membrane protein 2A (LMP2A) of Epstein-Barr virus. Our previous studies analyzing LMP2A function in vitro have suggested that Nedd4 family E3s regulate LMP2A function. To determine the role of Nedd4 family E3s in LMP2A B-cell signaling, LMP2A transgenic (LMP2A(+)) mice were crossed with mice with the Itch-deficient (Itch(-/-)) background. Itchy, a mouse homologue of human AIP4, is a Nedd4 family E3 and is also the most abundant Nedd4 family E3 found in LMP2A affinity precipitates from B cells. There were significantly fewer B-cell receptor-positive B cells in spleen and bone marrow B cells in LMP2A(+) Itch(-/-) mice than in LMP2A(+) mice. In addition, LMP2A(+) Itch(-/-) bone marrow B cells formed larger colonies in cultures treated with interleukin-7 (IL-7) than control bone marrow B cells did. Finally, there was a dramatic increase in tyrosine phosphorylation of LMP2A and Syk in IL-7-cultured LMP2A(+) Itch(-/-) B cells. These results indicate that Nedd4 family E3s, in particular Itchy, downmodulate LMP2A activity in B-cell signaling.     

IFN-beta mediates coordinate expression of antigen-processing genes in RSV-infected pulmonary epithelial cells

Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs) clear respiratory tract infections caused by the pneumovirus respiratory syncytial virus (RSV) and also mediate vaccine-induced pulmonary injury. Herein we examined the mechanism for RSV-induced MHC class I presentation. Like infectious viruses, conditioned medium from RSV-infected cells (RSV-CM) induces naive cells to coordinately express a gene cluster encoding the transporter associated with antigen presentation 1 (TAP1) and low molecular mass protein (LMP) 2 and LMP7. Neutralization of RSV-CM with antibodies to interferon (IFN)-beta largely blocked TAP1/LMP2/LMP7 expression, whereas anti-interleukin-1 antibodies were without effect, and recombinant IFN-beta increased TAP1/LMP2/LMP7 expression to levels produced by RSV-CM. LMP2, LMP7, and TAP1 expression were required for MHC class I upregulation because the irreversible proteasome inhibitor lactacystin or transfection with a competitive TAP1 inhibitor blocked inducible class I expression. We conclude that RSV infection coordinately increases MHC class I expression and proteasome activity through the paracrine action of IFN-beta to induce expression of the TAP1/LMP2/LMP7 locus, an event that may be important in the initiation of CTL-mediated lung injury.     

Bacterial lipopolysaccharide down-regulates expression of GTP cyclohydrolase I feedback regulatory protein.

GTP cyclohydrolase I feedback regulatory protein (GFRP) is a 9.7-kDa protein regulating GTP cyclohydrolase I activity in dependence of tetrahydrobiopterin and phenylalanine concentrations, thus enabling stimulation of tetrahydrobiopterin biosynthesis by phenylalanine to ensure its efficient metabolism by phenylalanine hydroxylase. Here, we were interested in regulation of GFRP expression by proinflammatory cytokines and stimuli, which are known to induce GTP cyclohydrolase I expression. Recombinant human GFRP stimulated recombinant human GTP cyclohydrolase I in the presence of phenylalanine and mediated feedback inhibition by tetrahydrobiopterin. Levels of GFRP mRNA in human myelomonocytoma (THP-1) cells remained unaltered by treatment of cells with interferon-gamma or interleukin-1beta, but were significantly down-regulated by bacterial lipopolysaccharide (LPS, 1 microg/ml), without or with cotreatment by interferon-gamma, which strongly up-regulated GTP cyclohydrolase I expression and activity. GFRP expression was also suppressed in human umbilical vein endothelial cells treated with 1 microg/ml LPS, as well as in rat tissues 7 h post intraperitoneal injection of 10 mg/kg LPS. THP-1 cells stimulated with interferon-gamma alone showed increased pteridine synthesis by addition of phenylalanine to the culture medium. Cells stimulated with interferon-gamma plus LPS, in contrast, showed phenylalanine-independent pteridine synthesis. These results demonstrate that LPS down-regulates expression of GFRP, thus rendering pteridine synthesis independent of metabolic control by phenylalanine.