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

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

Interferon-gamma inducible exchanges of 20S proteasome active site subunits: why?

When cells are stimulated with the cytokines IFN-gamma or TNF-alpha, the synthesis of three proteasome subunits LMP2 (beta1i), LMP7 (beta5i), and MECL-1 (beta2i) is induced. These subunits replace the three subunits delta (beta1), MB1 (beta5), and Z (beta2), which bear the catalytically active sites of the proteasome, during proteasome neosynthesis. The cytokine-induced exchanges of three active site subunits of a complex protease is unprecedented in biology and one may expect a strong functional driving force for this system to evolve. These cytokine-induced replacements of proteasome subunits are believed to favour the production of peptide ligands of major histocompatibility complex (MHC) class I molecules for the stimulation of cytotoxic T cells. Although the peptide production by constitutive proteasomes is able to maintain peptide-dependent MHC class I cell surface expression in the absence of LMP2 and LMP7, these subunits were recently shown to be pivotal for the generation or destruction of several unique epitopes. In this review we discuss the recent data on LMP2/LMP7/MECL-1-dependent epitope generation and the functions of each of these subunit exchanges. We propose that these subunit exchanges have evolved not only to optimize class I peptide loading but also to generate LMP2/LMP7/MECL-1-dependent epitopes in inflammatory sites which are not proteolytically generated in uninflamed tissues. This difference in epitope generation may serve to better stimulate T cells in the sites of an ongoing immune response and to avoid autoimmunity in uninflamed tissues.     

Identification and characterization of a beta proteasome subunit cluster in the Japanese pufferfish (Fugu rubripes)

The low molecular mass polypeptide (LMP2, LMP7, and MECL-1) genes code for beta-type subunits of the proteasome, a multimeric complex that degrades proteins into peptides as part of the MHC class I-mediated Ag-presenting pathway. These gene products are up-regulated in response to infection by IFN-gamma and replace the corresponding constitutively expressed subunits (X, Y, and Z) during the immune response. In humans, the LMP2 and LMP7 genes both reside within the class II region of the MHC (6p21.3), while MECL-1 is located at 16q22.1. In the present study, we have identified all three IFN-gamma-regulated beta-type proteasome subunits in Fugu, which are present as a cluster within the Fugu MHC class I region. We show that in this species, LMP7, LMP2, and MECL-1 are linked. Also within this cluster is an LMP2-like subunit (which seems specific to all teleosts tested to date) and a closely linked LMP7 pseudogene, indicating that within Fugu and potentially other teleosts, there has been an additional regional duplication involving these genes.     

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.     

Expression and regulation of interferon gamma-inducible proteasomal subunits LMP7 and LMP10 in the bovine corpus luteum

The proteasome is a large, polymeric protease complex responsible for intracellular protein degradation and generation of peptides that bind to class I major histocompatibility complex (MHC) molecules. Interferon gamma (INFgamma) induces expression of alternative proteasomal subunits that affect intracellular protein degradation, thereby changing the types of peptides that bind to class I MHC molecules. These alterations in class I MHC peptides can influence whether cells and tissues are tolerated by the immune system. Expression of two INFgamma-inducible proteasomal subunits, LMP7 and LMP10, in bovine luteal tissue was examined in this study. Northern analysis revealed the presence of mRNA encoding LMP7 and LMP10 in luteal tissue. Steady-state amounts of LMP7 mRNA did not change during the estrous cycle, but LMP10 mRNA was low in early corpus luteum (CL) and elevated in midcycle and late CL. Tumor necrosis factor alpha alone and in the presence of LH and/or prostaglandin F2alpha elevated steady-state amounts of LMP10 mRNA but did not affect LMP7 mRNA in cultured luteal cells. Immunohistochemistry revealed the presence of LMP10 primarily in small luteal cells. Numbers of LMP10-positive cells were lower in early CL than in midcycle and late CL. The finding that INFgamma-inducible proteasomal subunits are expressed in luteal tissue when the CL is fully functional was unexpected and suggests that proteasomes in luteal cells may generate peptides capable of stimulating a class I MHC-dependent inflammatory response.     

Regulation of immunoproteasome subunit expression in vivo following pathogenic fungal infection

The proteasome catalytic beta subunits LMP2, LMP7, and MECL-1 and two proteasome activator proteins, PA28 alpha and beta, are induced following exposure to IFN-gamma in vitro. Induction of these immunosubunits and the PA28 alpha/beta hetero-oligomer alters proteasome catalytic functions and specificity and enhances production of certain MHC class I epitopes. We sought to determine whether and to what extent proteasome subunit composition is regulated in vivo and to elucidate the mechanisms of such regulation. We analyzed basal expression levels of these inducible genes in normal, IFN-gamma-deficient, and Stat-1-deficient mice. Mice of all three genotypes display constitutive expression of the immunosubunits and PA28, demonstrating that basal expression in vivo is independent of endogenous IFN-gamma production. However, basal expression levels are reduced in Stat-1(-/-) mice, demonstrating a role for Stat-1 independent of IFN-gamma signaling. To demonstrate that IFN-gamma can induce these genes in vivo, mice were infected with Histoplasma capsulatum. Elevated expression of these genes followed the same time course as IFN-gamma expression in infected mice. IFN-gamma-deficient mice did not display elevated protein expression following infection, suggesting that other inflammatory cytokines produced in infected mice are unable to influence proteasome expression. Cytokines other than IFN-gamma also failed to influence proteasome gene expression in vitro in cell lines that had no basal expression of LMP2, LMP7, or MECL-1. Thus, both in vitro and in vivo data demonstrate that IFN-gamma is essential for up-regulation, but not constitutive expression, of immunoproteasome subunits in mice.     

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.     

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.     

Sequence information within proteasomal prosequences mediates efficient integration of beta-subunits into the 20 S proteasome complex.

The maturation of proteases is governed by prosequences. During the biogenesis of the highly oligomeric eukaryotic 20 S proteasome five different prosequence-containing subunits have to be integrated and processed either by autocatalysis or by neighbouring subunits. To analyse the functional impact of proteasomal prosequences during complex formation, the propeptide of the facultative subunit beta1i/LMP2 was truncated to nine amino acid residues or completely deleted. Additionally, the charged residues within the truncated beta1i/LMP2 version were replaced by neutral residues. While deletion did not affect subunit incorporation, the presence of charged residues within the truncated version of the LMP2 propeptide diminished incorporation efficiency, an effect that was restored upon replacement of the charged amino acids against neutral components. During immunoproteasome formation, incorporation and processing of inducible proteasome beta-subunits are cooperative processes. We demonstrate a linear correlation of the levels of beta2i/MECL1 and beta1i/LMP2 within 20 S proteasomes, suggesting a physical interaction to be the molecular basis for the biased incorporation of both subunits. In the absence of beta5i/LMP7, precursor complexes containing unprocessed beta1i/LMP2 accumulated. The contribution of beta5i/LMP7 on the cooperative formation of a homogeneous population of immunoproteasome is therefore most likely based on an acceleration of the beta1i/LMP2 and potentially of beta2i/MECL1 processing kinetics.     

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.     

Immune proteasomes in the development of the rat immune system

The dynamics of the expression of LMP7 and LMP2 proteasome subunits during embryonic and early postnatal development of rat spleen and liver was studied in comparison with the dynamics of chymotrypsin-like and caspase-like proteasome activities and expression of MHC (major histocompatibility complex) class I molecules. The distribution of LMP7 and LMP2 immune subunits in spleen and liver cells was also evaluated throughout development. The common tendency of both organs to increase the expression of both LMP7 and LMP2 subunits on the 21st postnatal day (P21) was found. However, the total proteasome level was shown to be constant. At certain developmental stages, the dynamics of immune subunits expression in the spleen and liver was different. While the gradual enhancement of both immune subunits was observed on P1, P18 and P21 in the spleen, the periods of gradual increase observed on E16 (the 16th embryonic day) and E18 gave way to a period of decrease in immune subunits on P5 in the liver. This level did not reliably change until P18 and increased on P21. The revealed changes were accompanied by an increase in chymotrypsin-like activity and a decrease in caspase-like activity in the spleen at P21 compared to the embryonic period. This indicates the increase in proteasome ability to form antigenic epitopes for MHC class I molecules. In the liver, both activities increased compared to the embryonic period by P21. The dynamics of caspase-like activity can be explained not only by the change of proteolytic constitutive and immune subunits, but also by additional regulatory mechanisms. Moreover, it was discovered that the increase in the expression of immune subunits during early spleen development is associated with the process of formation of white pulp by B- and T-lymphocytes enriched with immune subunits. In the liver, the increase in the level of immune subunits by P21 was also accompanied by an increase of their expression in hepatocytes. While the decrease of their level by P5 may be associated with the fact that the liver has lost its function as the primary lymphoid organ in the immune system by this time, as well as with the disappearance of B-lymphocytes enriched with immune proteasomes. In the spleen and the liver, MHC class I molecules were found during the periods of increased levels of proteasome immune subunits. On E21, the liver was enriched with neuronal nitric oxide synthase (nNOS); the level of nNOS decreased after birth and then increased by P18. This fact indicates the possibility of the induction of expression of the LMP7 and LMP2 immune subunits in hepatocytes via a signaling pathway involving nNOS. These results indicate that compared to the rat liver cells, splenic T cell immune response develops in rats starting around P19–P21. First, a T-area of white pulp is formed in the spleen during this period. Second, an increased level of immune proteasomes and MHC class I molecules in hepatocytes can ensure the formation of antigenic epitopes from foreign proteins and their delivery to the cell surface for subsequent presentation to cytotoxic T-lymphocytes.     

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.     

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.     

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.     

MHC class I-related antigen-processing machinery component defects in feline mammary carcinoma

Defects in HLA class I antigen-processing machinery (APM) component expression and/or function are frequent in human tumors. These defects may provide tumor cells with a mechanism to escape from recognition and destruction by HLA class I antigen-restricted, tumor antigen-specific cytotoxic T cells. However, expression and functional properties of MHC class I antigens and APM components in malignant cells in other animal species have been investigated to a limited extent. However, this information can contribute to our understanding of the mechanisms underlying the association of MHC class I antigen and APM component defects with malignant transformation of cells and to identify animal models to validate targeted therapies to correct these defects. To overcome this limitation in the present study, we have investigated the expression of the catalytic subunits of proteasome (Y, X, and Z) and of immunoproteasome (LMP2, LMP7, and LMP10) as well as of MHC class I heavy chain (HC) in 25 primary feline mammary carcinomas (FMCs) and in 23 matched healthy mammary tissues. We found a reduced expression of MHC class I HC and of LMP2 and LMP7 in tumors compared with normal tissues. Concordantly, proteasomal cleavage specificities in extracts from FMCs were different from those in healthy tissues. In addition, correlation analysis showed that LMP2 and LMP7 were concordantly expressed in FMCs, and their expression was significantly correlated with that of MHC class I HC. The abnormalities we have found in the APM in FMCs may cause a defective processing of some tumor antigens.     

Features of the immune proteasome expression in ascite Zajdela hepatoma after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis

The expression of the total proteasome pool, immune subunits LMP2 and LMP7, TAP1 and TAP2 transporters, and RT1A molecules of the major histocompatibility complex (MHC) class I in ascite Zajdela hepatoma cells was studied on the 10th day after implantation into Brattleboro rats with the hereditary defect in the synthesis of arginine-vasopressin (AVP) in the hypothalamus and WAG rats with normal AVP expression. Western-blot analysis revealed a threefold increase in the total number of proteasomes and immune subunit LMP2 and an eightfold increase in the immune subunits LMP7 in Zajdela hepatoma after its implantation in Brattleboro rats as compared with WAG rats. Differences in the expression of immune subunits LMP2 and LMP7 in Zajdela hepatoma in Brattleboro rats may contribute to different functions of these proteasomes, namely, the important role of the subunit LMP7 in antitumor immunity. Zajdela hepatoma growth in WAG rats was accompanied by a fall in both the total proteasome pool and immune proteasomes as compared with their content in Brattleboro rats, whose tumors regressed. The analysis of the content of peptide transporters TAP1 and TAP2 in Zajdela hepatoma implanted into Brattleboro and WAG rats showed their pronounced expression in tumor cells of both rat strains. In Zajdela hepatoma implanted into Brattleboro rats, a threefold increase in the basic molecule of MHC class I-RT1A was identified as compared with its expression in the tumor implanted to WAG rats. Furthermore, the content of CD8 and CD4 T-lymphocytes in the spleen of WAG and Brattleboro rats on the 10th day after implantation of Zajdela hepatoma was analyzed with flow cytometry. An increase in T-lymphocytes expressing the CD8 and CD4 antigens in the spleen of Brattleboro rats after implantation of the tumor as compared with WAG rats was shown. Increased numbers of both cytotoxic T lymphocytes and helper T-cells may facilitate tumor regression in Brattleboro rats. At the same time, a reduced number of subpopulations of T-lymphocytes in the spleen of WAG rats after implantation of hepatoma was accompanied by splenomegaly and growth of the tumor. Based on analysis of the data obtained it can be concluded that the deficiency of AVP in Brattleboro rats in Zajdela hepatoma leads to an increased expression of immune subunit LMP7 and basic molecules of MHC class I resulting in tumor immunogenicity and its elimination by the adaptive immune system.