Constitutive Proteasome Subunits Research Articles

Differential expression of constitutive and inducible proteasome subunits in human monocyte‐derived DC differentiated in the presence of IFN‐α or IL‐4

Several studies strongly suggest that DC differentiated in vitro in the presence of type I IFN acquire more potent immune stimulatory properties, compared with DC differentiated in vitro with IL-4. However, little is known about the molecular mechanisms underlying this phenomenon. To address this question, we compared the Ag-processing machinery (APM) profile in human DC grown in the presence of IFN-alpha ((IFN)DC) or IL-4 ((IL-4)DC). Using a panel of APM component-specific mAb in Western blot experiments, we found that (IFN)DC preferentially express inducible proteasome subunits (LMP2, LMP7, and MECL1) both at immature and mature stages. In contrast, immature (IL-4)DC co-express both constitutive (beta1, beta2, and beta5) and inducible subunits, as shown by Western blotting analysis. In addition, immature (IFN)DC express higher levels of TAP1, TAP2, calnexin, calreticulin, tapasin, and HLA class I molecules than (IL-4)DC. The different proteasome profiles of (IFN)DC and (IL-4)DC were associated with a greater ability of (IFN)DC to present an immunodominant epitope that requires LMP7 expression for its processing. In general, these data show the impact of cytokines on APM component expression and hence the Ag-processing ability of DC.

Functional alterations of the ubiquitin-proteasome system in motor neurons of a mouse model of familial amyotrophic lateral sclerosis†

In familial and sporadic amyotrophic lateral sclerosis (ALS) and in rodent models of the disease, alterations in the ubiquitin-proteasome system (UPS) may be responsible for the accumulation of potentially harmful ubiquitinated proteins, leading to motor neuron death. In the spinal cord of transgenic mice expressing the familial ALS superoxide dismutase 1 (SOD1) gene mutation G93A (SOD1G93A), we found a decrease in constitutive proteasome subunits during disease progression, as assessed by real-time PCR and immunohistochemistry. In parallel, an increased immunoproteasome expression was observed, which correlated with a local inflammatory response due to glial activation. These findings support the existence of proteasome modifications in ALS vulnerable tissues. To functionally investigate the UPS in ALS motor neurons in vivo, we crossed SOD1G93A mice with transgenic mice that express a fluorescently tagged reporter substrate of the UPS. In double-transgenic Ub(G76V)-GFP /SOD1G93A mice an increase in Ub(G76V)-GFP reporter, indicative of UPS impairment, was detectable in a few spinal motor neurons and not in reactive astrocytes or microglia, at symptomatic stage but not before symptoms onset. The levels of reporter transcript were unaltered, suggesting that the accumulation of Ub(G76V)-GFP was due to deficient reporter degradation. In some motor neurons the increase of Ub(G76V)-GFP was accompanied by the accumulation of ubiquitin and phosphorylated neurofilaments, both markers of ALS pathology. These data suggest that UPS impairment occurs in motor neurons of mutant SOD1-linked ALS mice and may play a role in the disease progression.

Automated Online Sequential Isotope Labeling for Protein Quantitation Applied to Proteasome Tissue-specific Diversity

Quantitation of protein abundance is a vital component in the proteomic analysis of biological systems, which can be achieved by differential stable isotopic labeling. To analyze tissue-derived samples, the isotopic labeling can be performed using chemical labeling of the peptides post-digestion. Standard chemical labeling procedures often require many manual sample handling steps, reducing the accuracy of measurements. Here, we describe a fully automated, online (in nanoLC columns), labeling procedure, which allows protein quantitation using differential isotopic dimethyl labeling of peptide N termini and lysine residues. We show that the method allows reliable quantitation over a wide dynamic range and can be used to quantify differential protein abundances in lysates and, more targeted, differences in composition between purified protein complexes. We apply the method to determine the differences in composition between bovine liver and spleen 20 S core proteasome complexes. We find that although all catalytically active immunoproteasome subunits were up-regulated in spleen (compared with liver), only one of the normal catalytic subunits was down-regulated, suggesting that the tissue-specific immunoproteasome assembly is more diverse than previously assumed.

Immunoproteasome-Selective Keto-Epoxide Inhibitors: Potential Therapeutic Agents for the Treatment of Hematological Disorders.

The clinical development of the dipeptide boronate bortezomib (Velcade™) has validated proteasome inhibition as a treatment for multiple myeloma and non-Hodgkin's lymphoma. In hematopoetically derived normal and malignant cells, the form of the proteasome expressed, termed the immunoproteasome, differs from that found in most tissues, referred to as the constitutive proteasome. Bortezomib inhibits the chymotrypsin-like activity of the immunoproteasome and the constitutive proteasome with equal potency and induces side effects such as peripheral neuropathy and gastrointestinal toxicity that may be related to proteasome inhibition in cells expressing the constitutive proteasome. This finding suggests that development of immunoproteasome-specific inhibitors would selectively target hematological malignances without affecting non-hematological cells. We have undertaken an effort to profile hematological tumor cell lines for immunoproteasome expression to determine which hematological cancers could be effectively targeted using an immunoproteasome selective inhibitor. We have observed that several cell lines derived from hematologic malignancies express predominantly the immunoproteasome while others express a mixture of both proteasome types. To develop selective inhibitors of the immunoproteasome, we generated analogs of PR-171, a peptide keto-epoxide that is currently being investigated in clinical trials for the treatment of myeloma and lymphoma. Purified 20S preparations were used to screen for compounds that selectively inhibit the chymotrypsin-like activity of the immunoproteasome (subunit LMP7) over the constitutive proteasome (subunit beta5). While the parental molecule PR-171 targets both forms of the proteasome with similar potency, several analogs have been identified with an IC50 below 50 nM on LMP7 and greater than 10-fold selectivity compared to beta5. To evaluate the immunoproteasome selectivity of our compounds in cells, an active site-specific ELISA was utilized. Cells expressing both constitutive and immunoproteasome were treated with the analogs and the occupancy of the LMP7 and beta5 active sites was determined. Several compounds retained their immunoproteasome selectivity in cells with an IC50 below 300 nM for LMP7 inhibition. In cell viability studies, some of these compounds demonstrated enhanced cytotoxicity in hematological tumor cell lines expressing the immunoproteasome as compared to solid tumor cell lines which express the constitutive proteasome. Finally, when pharmacodynamics was evaluated following intravenous administration to mice, LMP7 selectivity was retained in tissues expressing both the immunoproteasome and constitutive proteasome. The in vivo anti-tumor properties and toxicity profiles of the immunoproteasome-selective inhibitors are currently being evaluated.

A Proteasome Specific Binding Assay for Quantitation of Constitutive and Immunoproteasome Active Sites.

The ubiquitin-proteasome pathway constitutes a major intracellular system for protein degradation. Substrates for this pathway include misfolded or unassembled proteins as well as short-lived regulatory proteins that play key roles in signaling and proliferative pathways. The majority of cell types express the standard, or “constitutive”, form of the proteasome, while cells of the immune system also express the immunoproteasome, a form of the proteasome that contributes to class I major histocompatibility complex restricted antigen processing. Non-immune cells can also express immunoproteasome in response to interferon gamma exposure. The immunoproteasome retains the same structural subunits as the constitutive proteasome but has three different catalytic subunits. The catalytic activities of both forms of the proteasome have been traditionally characterized with purified enzyme preparations and fluorogenic peptide substrates. Such fluorogenic peptide substrates suffer from two characteristics that limit their utility in measuring proteasome activities in complex cell or tissue lysates: 1) they cannot distinguish proteasome activities from other proteolytic activities within the lysate; and 2) they can not distinguish between constitutive and immunoproteasome activities. We have developed an ELISA-based proteasome-specific binding (PSB) assay that can detect and quantify the chymotryptic-like proteasome active sites of the beta-5 constitutive proteasome subunit and the LMP7 immunoproteasome subunit. The assay utilizes a biotin-modified peptide epoxyketone probe that covalently and irreversibly interacts with the active site threonine present in catalytic proteasome subunits. Once bound to the probe, the labeled subunits are recovered on streptavidin-conjugated beads and detected with subunit-specific antibodies. The PSB assay is both quantitative and sensitive. We have demonstrated that the assay is capable of measuring constitutive proteasome and immunoproteasome binding activity in human whole blood and peripheral blood mononuclear cell preparations, respectively. In experiments with the epoxyketone-based proteasome inhibitor PR-171, the dose response for inhibition of the PSB assay is equivalent to that measured with a conventional fluorogenic peptide proteasome substrate. In addition, the PSB assay can effectively measure the level of PR-171 mediated inhibition of both the constitutive and immunoproteasome in the RPMI-8226 multiple myeloma cell line that co-expresses both proteasome types. Thus, the PSB assay overcomes the limitations of conventional fluorogenic substrate-based proteasome activity assays when applied to cell or tissue lysates that contain multiple proteolytic activities or mixtures of constitutive and immunoproteasomes. Potential applications of the PSB assay include the measurement of the pharmacodynamic response to proteasome inhibitors and the evaluation of constitutive vs. immunoproteasome selectivity of inhibitors both in vitro and in vivo.

Development and characterization of human constitutive proteasome and immunoproteasome subunit‐specific monoclonal antibodies

Delta (Y), MB1 (X), and Z are the three catalytic beta-subunits located in the inner rings of the constitutive proteasome, an intracellular multicatalytic complex responsible for the generation of peptides presented by human leukocyte antigen (HLA) class I antigens to T cells. When cells are incubated with interferon-gamma, delta (Y), MB1 (X), and Z are replaced by LMP2, LMP7, and LMP10, respectively, leading to the expression of immunoproteasome which generates peptides with increased affinity for HLA class I antigens. The characterization of the expression of constitutive proteasome and immunoproteasome subunits in cells, normal tissues, and malignant lesions has been hampered by the lack or limited availability of constitutive proteasome and immunoproteasome subunit-specific monoclonal antibodies (mAbs), which are suitable for immunohistochemical staining. To overcome this limitation, we generated human delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10-specific mAb-secreting hybridomas from BALB/c mice immunized with peptides and recombinant fusion proteins. The mAbs SY-5, SJJ-3, NB-1, SY-1, HB-2, and TO-7 were shown to be specific for delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10, respectively, as they react specifically with the corresponding molecules when tested with a human B lymphoid LG2 cell lysate in Western blotting and with the peptide derived from each molecule in enzyme-linked immunosorbent assay. The reactivity of the six mAbs with the corresponding intracellular antigens resulted in intracellular staining when the mAbs were tested with microwave-treated and saponin-permeabilized cells in indirect immunofluorescence and with formalin-fixed, paraffin-embedded tissue sections in immunohistochemical reactions. These results suggest that the constitutive proteasome and immunoproteasome subunit-specific mAbs we have developed are useful probes to characterize the expression of proteasome subunits in normal tissues and in pathological lesions.

T Cell Activation by t(9;22) Acute Lymphoblastic Leukemia-Derived Dendritic-Like Cells Is Associated with Increased Expression of Antigen Processing Machinery Components.

We have recently shown that dendritic-like cells derived from t(9;22) acute lymphoblastic leukemia (ALL) cells can activate T cells, while the original unmodified leukemic blasts cannot. To define the molecular basis of this functional difference, we have compared the level of antigen processing machinery components in unmodified blasts and ALL-derived dendritic-like cells, since they play a major role in antigen presentation. Six patient samples and one t(9;22) ALL cell line (Z119) were studied.Dendritic-like cells were generated by in vitro culture with CD40 ligand, interleukin (IL)-1β, IL-3, IL-7, stem cell factor and tumor necrosis factor-α for six days. Maturation was verified by morphology and CD80/CD86 expression. Cells were studied for HLA class I and class II antigen expression. Furthermore, antigen processing machinery component expression was analyzed by intracellular staining with a unique panel of monoclonal antibodies which recognize the constitutive proteasome (Z, MB1, delta) and immunoproteasome (LMP2, LMP7, LMP10) subunits, the transporter subunits TAP 1 and 2 and the chaperones calnexin, ERp57, calreticulin and tapasin. HLA Class I antigen and HLA-DR/DQ/DP antigen expression was significantly up-regulated on the dendritic-like cells. LMP2, LMP7 and tapasin expression was significantly up-regulated in all t(9;22) ALL-derived dendritic-like cells, in comparison to the unmodified blasts. No significant difference was detected in the expression of the other antigen processing machinery components. These results suggest that T cell activation by t(9;22) ALL-derived dendritic-like cells is associated with increased expression of some, but not all, of the antigen processing machinery components. Whether t(9;22) ALL-derived dendritic-like cells can be used as a cellular vaccine for adoptive immunotherapy of ALL is being investigated.

Coordinate downregulation of multiple MHC class I antigen processing genes in chemical-induced murine tumor cell lines of distinct origin.

In murine tumor cell lines, downregulation of MHC class I surface expression has been frequently detected, but the underlying molecular mechanisms of such deficiencies have not been defined. In this study, murine tumor cell lines of different histology derived from spontaneous or from chemical-induced tumors were analyzed for the expression of multiple components of the major histocompatibility complex (MHC) class I antigen-processing machinery (APM), including the peptide transporter TAP, the interferon (IFN)-gamma inducible proteasome subunits and several chaperones. The tumor cell lines analyzed demonstrated a heterogeneous expression pattern of various APM components. In comparison to control cells an impaired coordinated expression of at least three APM components was detected. In particular, extensive APM deficiencies were found in cell lines derived from chemical-induced tumors. A strong coordinated downregulation of expression and/or function of TAP, the low molecular weight proteins (LMP) subunits, the proteasome activator PA28 and/or tapasin was found in 5 of 10 tumor cells, which was associated with impaired MHC class I surface expression. In contrast, the expression of beta2-microglobulin (beta2-m), PA28beta, the constitutive proteasome subunits X, Y, Z and of the chaperones calnexin, calreticulin, ER60 and phospho disulfide isomerase (PDI) was unaltered or only weakly decreased. The deficient expression of APM components could be corrected by IFN-gamma treatment, which also reconstituted MHC class I surface expression. However, impaired expression of APM molecules appears not to be the only cause of abnormal MHC class I expression, since it could neither be corrected by the addition of exogeneous MHC class I binding peptides nor by incubation at low temperature. These results suggest that one major mechanism of murine tumor cells, in particular chemical-induced tumors, to evade the immune system is the combined dysregulation of various APM components and other factors, which still have to be identified.

Role of Proteasomes in Antigen Presentation

Recent studies have demonstrated that the proteasome, in addition to functioning in the complete degradation of cell proteins, is the source of most antigenic peptides presented to the immune system on major histocompatibility complex (MHC)-class I molecules. In this process, intracellular and viral proteins are degraded in the cytosol to 8- to 9-amino acid fragments, which are then transported into the endoplasmic reticulum, where they become associated with MHC-class I molecules and are thus delivered to the cell surface. A variety of evidence has shown that the proteasome and ATP-ubiquitin-dependent pathway are critical in this process: (1) In cells, selective inhibitors of proteasome function inhibit the bulk of protein degradation and thus prevent the generation of peptides necessary for class I presentation and the appearance of MHC on the cell surface. (2) Mutations that block ubiquitin conjugation prevent the generation of an antigenic peptide. (3) Modifications that lead to rapid degradation of a protein by the ubiquitin pathway enhance antigen presentation. (4) gamma-Interferon (gamma-IFN) induces new proteasome subunits, LMP2 and LMP7, encoded in the MHC region that are incorporated in place of constitutive proteasome subunits. Their incorporation does not affect rates of protein breakdown but causes changes in peptidase activities, i.e. they increase rates of cleavage after basic and hydrophobic residues and decrease cleavage after acidic residues. Transfections of cells with LMP2 or LMP7 cause similar changes in these peptidase activities as are caused by gamma-IFN. These modifications in peptidase activities should enhance the production of those types of peptides which are preferentially transported into endoplasmic reticulum and selectively bound to MHC-class I molecules.

Carcinoma of the Stomach

Salinosporamide A (NPI-0052, marizomib) is a naturally occurring proteasome inhibitor derived from the marine actinobacterium Salinispora tropica, and represents a promising clinical agent in the treatment of hematologic malignancies. Recently, these actinobacteria were shown to harbor self-resistance properties to salinosporamide A by expressing redundant catalytically active mutants of the 20S proteasome β-subunit, reminiscent of PSMB5 mutations identified in cancer cells with acquired resistance to the founding proteasome inhibitor bortezomib (BTZ). Here, we assessed the growth inhibitory potential of salinosporamide A in human acute lymphocytic leukemia CCRF-CEM cells, and its 10-fold (CEM/BTZ7) and 123-fold (CEM/BTZ200) bortezomib-resistant sublines harboring PSMB5 mutations. Parental cells displayed sensitivity to salinosporamide A (IC50 = 5.1 nM), whereas their bortezomib-resistant sublines were 9- and 17-fold cross-resistant to salinosporamide A, respectively. Notably, combination experiments of salinosporamide A and bortezomib showed synergistic activity in CEM/BTZ200 cells. CEM cells gradually exposed to 20 nM salinosporamide A (CEM/S20) displayed stable 5-fold acquired resistance to salinosporamide A and were 3-fold cross-resistant to bortezomib. Consistent with the acquisition of a PSMB5 point mutation (M45V) in CEM/S20 cells, salinosporamide A displayed a markedly impaired capacity to inhibit β5-associated catalytic activity. Last, compared with parental CEM cells, CEM/S20 cells exhibited up to 2.5-fold upregulation of constitutive proteasome subunits, while retaining unaltered immunoproteasome subunit expression. In conclusion, salinosporamide A displayed potent antileukemic activity against bortezomib-resistant leukemia cells. β-Subunit point mutations as a common feature of acquired resistance to salinosporamide A and bortezomib in hematologic cells and S. tropica suggest an evolutionarily conserved mechanism of resistance to proteasome inhibitors.