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.     

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.     

Immunoproteasome Deficiency Protects in the Retina after Optic Nerve Crush

The immunoproteasome is upregulated by disease, oxidative stress, and inflammatory cytokines, suggesting an expanded role for the immunoproteasome in stress signaling that goes beyond its canonical role in generating peptides for antigen presentation. The signaling pathways that are regulated by the immunoproteasome remain elusive. However, previous studies suggest a role for the immunoproteasome in the regulation of PTEN and NF-κB signaling. One well-known pathway upstream of NF-κB and downstream of PTEN is the Akt signaling pathway, which is responsible for mediating cellular survival and is modulated after optic nerve crush (ONC). This study investigated the role of retinal immunoproteasome after injury induced by ONC, focusing on the Akt cell survival pathway. Retinas or retinal pigment epithelial (RPE) cells from wild type (WT) and knockout (KO) mice lacking either one (LMP2) or two (LMP7 and MECL-1) catalytic subunits of the immunoproteasome were utilized in this study. We show that mRNA and protein levels of the immunoproteasome subunits are significantly upregulated in WT retinas following ONC. Mice lacking the immunoproteasome subunits show either a delayed or dampened apoptotic response as well as altered Akt signaling, compared to WT mice after ONC. Treatment of the RPE cells with insulin growth factor-1 (IGF-1) to stimulate Akt signaling confirmed that the immunoproteasome modulates this pathway, and most likely modulates parallel pathways as well. This study links the inducible expression of the immunoproteasome following retinal injury to Akt signaling, which is important in many disease pathways.     

Evolution of the proteasome components

 A phylogenetic analysis of proteasome subunits revealed two major families (α and β) which originated by an ancient gene duplication prior to the divergence of archaebacteria and eukaryotes. Numerous gene duplications have subsequently occurred in eukaryotes; at least nine of these duplications were shown to have occurred prior to the divergence of animals and fungi. In mammals, two genes encoding proteasome subunits (LMP2 and LMP7) are located in the major histocompatibility complex (MHC) region and play a specific role in generation of peptides for presentation by class I MHC molecules. Phylogenetic analysis of LMP7 and related sequences from mammals and lower vertebrates indicated that this locus arose by gene duplication prior to the divergence of jawed and jawless vertebrates; the time of this duplication was estimated to have been about 600 million years ago. The evolutionary history of the proteasome subunits provides support for a model of the evolution of new gene function postulating that, after gene duplication, the proteins encoded by daughter loci can adapt to specialized functions previously performed by the product of a single generalized ancestral locus. Received: 19 August 1996 / Revised: 24 December 1996     

Role of immunoproteasome catalytic subunits in the immune response to hepatitis B virus

Inhibition of hepatitis B virus (HBV) replication and viral clearance from an infected host requires both the innate and adaptive immune responses. Expression of interferon (IFN)-inducible proteasome catalytic and regulatory subunits correlates with the IFN-alpha/beta- and IFN-gamma-mediated noncytopathic inhibition of HBV in transgenic mice and hepatocytes, as well as with clearance of the virus in acutely infected chimpanzees. The immunoproteasome catalytic subunits LMP2 and LMP7 alter proteasome specificity and influence the pool of peptides available for presentation by major histocompatibility complex class I molecules. We found that these subunits influenced both the magnitude and specificity of the CD8 T-cell response to the HBV polymerase and envelope proteins in immunized HLA-A2-transgenic mice. We also examined the role of LMP2 and LMP7 in the IFN-alpha/beta- and IFN-gamma-mediated inhibition of virus replication using HBV transgenic mice and found that they do not play a direct role in this process. These results demonstrate the ability of the IFN-induced proteasome catalytic subunits to shape the HBV-specific CD8 T-cell response and thus potentially influence the progression of infection to acute or chronic disease. In addition, these studies identify a potential key role for IFN in regulating the adaptive immune response to HBV through alterations in viral antigen processing.     

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.     

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.     

Purification and separation of the 20S immunoproteasome from the constitutive proteasome and identification of the subunits by LC–MS

The proteasome is a multicatalytic protease complex present in all eukaryotic cells, which plays a critical role in regulating essential cellular processes. During the immune response to pathogens, stimulation by γ interferon induces the production of a special form of proteasome, the immunoproteasome. Inappropriate increase of proteosomal activity has been linked to inflammatory and autoimmune diseases. Selective inhibition of the immunoproteasome specific LMP7 subunit was shown to block inflammatory cytokine secretion in human PBMC, thus making the immunoproteasome an interesting target to fight autoimmune diseases. This paper describes a method for purification and separation of the 20S immunoproteasomes from the constitutive proteasome, which is ubiquitously present in all cells, based on hydrophobic interaction chromatography. The purified immunoproteasome showed several bands, between 20–30 kDa, when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The purified proteasome complexes had a molecular mass of approximately 700 kDa as estimated by gel filtration. Identification of the catalytic subunits in the immunoproteasomes was performed in Western blot with antibodies directed specifically against either the constitutive or the immunoproteasome subunits. The purified immunoproteasome possessed all three protease activities associated with the proteasome complex. LC/MS analysis confirmed the presence of the three immunoproteasome catalytic subunits in the purified immunoproteasome.     

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.     

Beta 2 subunit propeptides influence cooperative proteasome assembly

Vertebrate proteasomes are structurally heterogeneous, consisting of both "constitutive" (or "standard") proteasomes and "immunoproteasomes." Constitutive proteasomes contain three ubiquitously expressed catalytic subunits, Delta (beta 1), Z (beta 2), and X (beta 5), whereas immunoproteasomes contain three interferon-gamma-inducible catalytic subunits, LMP2 (beta 1i), MECL (beta 2i), and LMP7 (beta 5i). We recently have demonstrated that proteasome assembly is biased to promote immunoproteasome homogeneity when both types of catalytic subunits are expressed in the same cell. This cooperative assembly is due in part to differences between the LMP7 (beta 5i) and X (beta 5) propeptides. In the current study we demonstrate that differences between the MECL (beta 2i) and Z (beta2) propeptides also influence cooperative assembly. Specifically, replacing the MECL propeptide with that of Z enables MECL incorporation into otherwise constitutive (Delta(+)/X(+)) proteasomes and facilitates X incorporation into otherwise immunoproteasomes (MECL(+)/LMP2(+)). We also show, using MECL(-/-) mice, that LMP2 incorporation does not require MECL, in contrast with previous suggestions that their incorporation is mutually codependent. These results enable us to refine our model for cooperative proteasome assembly by determining which combinations of inducible and constitutive subunits are favored over others, and we propose a mechanism for how propeptides mediate cooperative assembly.     

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.     

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.     

Novel propeptide function in 20 S proteasome assembly influences beta subunit composition

The assembly of eukaryotic 20 S proteasomes involves the formation of half-proteasomes where precursor beta-type subunits gather in position on an alpha-subunit ring, followed by the association of two half-proteasomes and beta-subunit processing. In vertebrates three additional beta-subunits (beta1i/LMP2, beta2i/MECL1, and beta5i/LMP7) can be synthesized and substituted for constitutive homologues (beta1/delta, beta2/Z, and beta5/X) to yield immunoproteasomes, which are important for generating certain antigenic peptides. We have shown previously that when all six beta-subunits are present, cooperative assembly mechanisms limit the diversity of proteasome populations. Specifically, LMP7 is incorporated preferentially over X into preproteasomes containing LMP2 and MECL1. We show here that the LMP7 propeptide is responsible for this preferential incorporation, and it also enables LMP7 to incorporate into proteasomes containing delta and Z. In contrast, the X propeptide restricts incorporation to proteasomes with delta and Z. Furthermore, we demonstrate that the LMP7 propeptide can function in trans when expressed on LMP2, and that its NH(2)-terminal and mid-regions are particularly critical for function. In addition to identifying a novel propeptide function, our results raise the possibility that one consequence of LMP7 incorporation into both immunoproteasomes and delta/Z proteasomes may be to increase the diversity of antigenic peptides that can be generated.     

Immunoproteasome-deficient mice mount largely normal CD8+ T cell responses to lymphocytic choriomeningitis virus infection and DNA vaccination

During viral infection, constitutive proteasomes are largely replaced by immunoproteasomes, which display distinct cleavage specificities, resulting in different populations of potential CD8(+) T cell epitope peptides. Immunoproteasomes are believed to be important for the generation of many viral CD8(+) T cell epitopes and have been implicated in shaping the immunodominance hierarchies of CD8(+) T cell responses to influenza virus infection. However, it remains unclear whether these conclusions are generally applicable. In this study we investigated the CD8(+) T cell responses to lymphocytic choriomeningitis virus infection and DNA immunization in wild-type mice and in mice lacking the immunoproteasome subunits LMP2 or LMP7. Although the total number of virus-specific cells was lower in LMP2 knockout mice, consistent with their having lower numbers of naive cells before infection, the kinetics of virus clearance were similar in all three mouse strains, and LMP-deficient mice mounted strong primary and secondary lymphocytic choriomeningitis virus-specific CD8(+) T cell responses. Furthermore, the immunodominance hierarchy of the four investigated epitopes (nuclear protein 396 (NP(396)) > gp33 > gp276 > NP(205)) was well maintained. We observed a slight reduction in the NP(205)-specific response in LMP2-deficient mice, but this had no demonstrable biological consequence. DNA vaccination of LMP2- and LMP7-deficient mice induced CD8(+) T cell responses that were slightly lower than, although not significantly different from, those induced in wild-type mice. Taken together, our results challenge the notion that immunoproteasomes are generally needed for effective antiviral CD8(+) T cell responses and for the shaping of immunodominance hierarchies. We conclude that the immunoproteasome may affect T cell responses to only a limited number of viral epitopes, and we propose that its main biological function may lie elsewhere.     

Suppression of the Macrophage Proteasome by Ethanol Impairs MHC Class I Antigen Processing and Presentation

Alcohol binge-drinking (acute ethanol consumption) is immunosuppressive and alters both the innate and adaptive arms of the immune system. Antigen presentation by macrophages (and other antigen presenting cells) represents an important function of the innate immune system that, in part, determines the outcome of the host immune response. Ethanol has been shown to suppress antigen presentation in antigen presenting cells though mechanisms of this impairment are not well understood. The constitutive and immunoproteasomes are important components of the cellular proteolytic machinery responsible for the initial steps critical to the generation of MHC Class I peptides for antigen presentation. In this study, we used an in-vitro cell culture model of acute alcohol exposure to study the effect of ethanol on the proteasome function in RAW 264.7 cells. Additionally, primary murine peritoneal macrophages obtained by peritoneal lavage from C57BL/6 mice were used to confirm our cell culture findings. We demonstrate that ethanol impairs proteasome function in peritoneal macrophages through suppression of chymotrypsin-like (Cht-L) proteasome activity as well as composition of the immunoproteasome subunit LMP7. Using primary murine peritoneal macrophages, we have further demonstrated that, ethanol-induced impairment of the proteasome function suppresses processing of antigenic proteins and peptides by the macrophage and in turn suppresses the presentation of these antigens to cells of adaptive immunity. The results of this study provide an important mechanism to explain the immunosuppressive effects of acute ethanol exposure.     

Studies on immunoproteasome in human liver. Part I: Absence in fetuses, presence in normal subjects, and increased levels in chronic active hepatitis and cirrhosis

Despite the central role of proteasomes in relevant physiological pathways and pathological processes, this topic is unexpectedly largely unexplored in human liver. Here we present data on the presence of proteasome and immunoproteasome in human livers from normal adults, fetuses and patients affected by major hepatic diseases such as cirrhosis and chronic active hepatitis. Immunohistochemistry for constitutive (α4 and β1) and inducible (LMP2 and LMP7) proteasome subunits, and for the PA28αβ regulator, was performed in liver samples from 38 normal subjects, 6 fetuses, 2 pediatric cases, and 19 pathological cases (10 chronic active hepatitis and 9 cirrhosis). The immunohistochemical data have been validated and quantified by Western blotting analysis. The most striking result we found was the concomitant presence in hepatocyte cytoplasm of all healthy subjects, including the pediatric cases, of constitutive proteasome and immunoproteasome subunits, as well as PA28αβ. At variance, immunoproteasome was not present in hepatocytes from fetuses, while a strong cytoplasmic and nuclear positivity for LMP2 and LMP7 was found in pathological samples, directly correlated to the histopathological grade of inflammation. At variance from other organs such as the brain, immunoproteasome is present in livers from normal adult and pediatric cases, in apparent absence of pathological processes, suggesting the presence of a peculiar regulation of the proteasome/immunoproteasome system, likely related to the physiological stimuli derived from the gut microbiota after birth. Other inflammatory stimuli contribute in inducing high levels of immunoproteasome in pathological conditions, where its role deserve further attention.