Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The proteasome system represents a major source of HLA class I- presented peptides exposed to CTLs. Stimulation of cells with IFN-gamma instantly induces the expression of the proteasome immunosubunits as well as the proteasome activator PA28. These proteins have been shown to optimize class I antigen presentation of several viral CTL epitopes; however, their contribution to tumor antigen processing remains poorly understood. Here, we analyzed the generation of an HLA-A*0201-presented epitope derived from the melanoma antigen tyrosinase-related protein 2 (TRP2). Melanoma cells that lacked the IFN-gamma-inducible proteasome activator PA28 and immunoproteasomes did not display the TRP2(360-368) epitope to specific CTLs. Our experiments demonstrate that epitope presentation correlated with the presence of PA28 and could be completely rescued by restoration of PA28 expression. In vitro digestion of TRP2 polypeptides with 20S proteasomes confirmed that PA28 is essential for epitope liberation. Thus, our experiments indicate that PA28 provides the threshold for CTL recognition of this epitope. Importantly, processing of a second TRP2-derived epitope, TRP2(288-296), was diminished in IFN-gamma-treated cells, even in the absence of immunoproteasome up-regulation. Therefore, the reported IFN-gamma-induced self-regulation of epitopes may not necessarily be a consequence of immunoproteasomes as suggested previously.
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PMID:Expression of the proteasome activator PA28 rescues the presentation of a cytotoxic T lymphocyte epitope on melanoma cells. 1201 67

EBV-infected cells and EBV-associated tumors may evade CTL recognition by defective antigen processing, resulting in poor presentation of CTL epitopes. Since the proteasome is the major source of MHC class I-presented peptides, we analyzed the effect of proteasome inhibitors on the expression of surface HLA class I and the generation of EBV-derived CTL epitopes presented by the HLA-A2 and HLA-A11 alleles. Treatment with covalent and reversible inhibitors of the proteasome partially reduced the total and allele-specific expression of surface HLA class I in EBV-carrying LCLs. HLA-A2 expression was also decreased by treatment with leupeptin and bestatin, while HLA-A11 expression was affected by treatment with phenanthroline. Despite their general inhibitory effect on HLA class I expression, all proteasome inhibitors tested enhanced the presentation of 2 subdominant HLA-A2 epitopes from EBV LMP1 and LMP2, while the presentation of the immunodominant HLA-A11-restricted epitope from EBNA4 was inhibited by MG132 and lactacystin and increased by ZL(3)VS. Treatment with ZL(3)VS restored the presentation of endogenously expressed EBNA4 in 1 HLA-A11-positive BL cell line. These findings suggest that specific inhibitors of the proteasome may be used to increase the antigenicity of virus-infected and malignant cells that are per se inefficient at generating particular CTL target epitopes.
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PMID:Proteasome inhibitors reconstitute the presentation of cytotoxic T-cell epitopes in Epstein-Barr virus-associated tumors. 1223 93

Treatment of melanoma cell lines with IFN-gamma induces the switch from proteasome (PS) to immunoproteasome (iPS). This finding has profound implications for the immunobiology of melanoma cells since certain peptides (such as Melan-A(mart1)(27-35)) are cleaved differently by iPS, thus implying a different ability to be presented by HLA class I molecules. IFN-alpha is a cytokine not only produced during infectious diseases, but also used in the treatment of certain cancers. Nevertheless, the effects of IFN-alpha on the switch of PS to iPS are largely unknown. A comparison of the effect of both IFN-alpha and IFN-gamma was thus carried out on melanoma cell lines. RT-PCR showed that mRNA for iPS subunits (i.e. LMP-2, LMP-7 and MECL-1) was detectable both in untreated and IFN-treated melanoma cells. Immunoblotting analysis revealed that while IFN-gamma was able to consistently induce the switch from PS to iPS, IFN-alpha treatment did not, possibly due to post-transcriptional event(s) blocking the expression of iPS-specific subunits. Finally, Melan-A(mart1)(27-35) peptide was found only in the HPLC-MS spectra from both untreated and IFN-alpha-treated cells, but not upon IFN-gamma treatment. Altogether, these data demonstrate that IFN-alpha does not induce the switch from PS to iPS.
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PMID:IFN-alpha mediates the up-regulation of HLA class I on melanoma cells without switching proteasome to immunoproteasome. 1464 50

Malignant transformation of breast epithelia is frequently associated with an altered expression of MHC products and of antigen processing molecular machinery. The consequent impairment of tumor immune recognition is thought to confer to tumor cells a selective advantage with respect to survival and metastatization. In order to understand if metastatic breast cancer lesions might be associated with a defective proteasome subunit expression that, in turn, might limit the peptide availability and prevent stable cell surface HLA class I-tumor antigen expression, we studied by immunostaining the expression of beta2-microglobulin, HLA class I antigens and proteasome subunits LMP-2 and LMP-10 in 35 matched primary and metastatic human breast carcinoma lesions. Overall, we found a downregulation of LMP-2 in 51.4% of the lesions, of LPM-10 in 45.7% of the lesions, of HLA class I heavy chain in 40.0% of the lesions, while beta2-microglobulin was downregulated in 25.7% of the lesions studied. In most primary and metastatic lesions the downmodulation of each antigen examined was coordinated. In the cases where a selective downmodulation of antigens was observed in the primary or in the metastatic lesion (with the exception of beta2-microglobulin), it was rather observed in the primary lesions. However, LMP-10 showed a significant selective downmodulation in the metastases as well. Antigen downmodulation does not appear therefore to represent a strategy for the primary tumor to metastasize successfully.
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PMID:Expression of HLA class I antigen and proteasome subunits LMP-2 and LMP-10 in primary vs. metastatic breast carcinoma lesions. 1554 99

Type 1 diabetes is thought to result from the destruction of beta-cells by autoantigen-specific T-cells. Observations in the NOD mouse model suggest that CD8+ cytotoxic T-cells play an essential role in both the initial triggering of insulitis and its destructive phase. However, little is known about the epitopes derived from human beta-cell autoantigens and presented by HLA class I molecules. We used a novel reverse immunology approach to identify HLA-A2-restricted, naturally processed epitopes derived from proinsulin, an autoantigen likely to play an important role in the pathogenesis of type 1 diabetes. Recombinant human proinsulin was digested with purified proteasome complexes to establish an inventory of potential COOH-terminals of HLA class I-presented epitopes. Cleavage data were then combined with epitope predictions based on the SYFPEITHI and BIMAS algorithms to select 10 candidate epitopes; 7 of these, including 3 with a sequence identical to murine proinsulin, were immunogenic in HLA-A2 transgenic mice. Moreover, six of six tested peptides were processed and presented by proinsulin-expressing cells. These results demonstrate the power of reverse immunology approaches. Moreover, the novel epitopes may be of significant interest in monitoring autoreactive T-cells in type 1 diabetes.
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PMID:Identification of naturally processed HLA-A2--restricted proinsulin epitopes by reverse immunology. 1598 6

Proinsulin is a key autoantigen in type 1 diabetes. Evidence in the mouse has underscored the importance of the insulin B chain region in autoimmunity to pancreatic beta cells. In man, a majority of proteasome cleavage sites are predicted by proteasome cleavage algorithms within this region. To study CD8+ T cell responses to the insulin B chain and adjacent C peptide, we selected 8- to 11-mer peptides according to proteasome cleavage patterns obtained by digestion of two peptides covering proinsulin residues 28 to 64. We studied their binding to purified HLA class I molecules and their recognition by T cells from diabetic patients. Peripheral blood mononuclear cells from 17 of 19 recent-onset and 12 of 13 long-standing type 1 diabetic patients produced IFN-gamma in response to proinsulin peptides as shown by using an ELISPOT assay. In most patients, the response was against several class I-restricted peptides. Nine peptides were recognized within the proinsulin region covering residues 34 to 61. Four yielded a high frequency of recognition in HLA-A1 and -B8 patients. Three peptides located in the proinsulin region 41-51 were shown to bind several HLA molecules and to be recognized in a high percentage of diabetic patients.
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PMID:Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic patients. 1603 Jan 47

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.
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PMID:Development and characterization of human constitutive proteasome and immunoproteasome subunit-specific monoclonal antibodies. 1610 29

Myeloma vaccines, based on dendritic cells pulsed with idiotype or tumor lysate, have been met with limited success, probably in part due to insufficient cross-priming of myeloma antigens. A powerful method to introduce myeloma-associated antigens into the cytosol of dendritic cells is protein transduction, a process by which proteins fused with a protein transduction domain (PTD) freely traverse membrane barriers. NY-ESO-1, an immunogenic antigen by itself highly expressed in 60% of high-risk myeloma patients, was purified to near homogeneity both alone and as a recombinant fusion protein with a PTD, derived from HIV-Tat. Efficient entry of PTD-NY-ESO-1 into dendritic cells, confirmed by microscopy, Western blotting, and intracellular flow cytometry, was achieved without affecting dendritic cell phenotype. Experiments with amiloride, which inhibits endocytosis, and N-acetyl-l-leucinyl-l-norleucinal, a proteasome inhibitor, confirmed that PTD-NY-ESO-1 entered dendritic cells by protein transduction and was degraded by the proteasome. Tetramer analysis indicated superior generation of HLA-A2.1, CD8+ T lymphocytes specific for NY-ESO-1(157-165) with PTD-NY-ESO-1 compared with NY-ESO-1 control protein (44% versus 2%, respectively). NY-ESO-1-specific T lymphocytes generated with PTD-NY-ESO-1 secreted IFN-gamma indicative of a Tc1-type cytokine response. Thus, PTD-NY-ESO-1 accesses the cytoplasm by protein transduction, is processed by the proteasome, and NY-ESO-1 peptides presented by HLA class I elicit NY-ESO-1-specific T lymphocytes.
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PMID:Protein transduction of dendritic cells for NY-ESO-1-based immunotherapy of myeloma. 1626 30

For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.
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PMID:BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes. 1693 47

Type 1 diabetes mellitus (T1DM) results from the destruction of beta cells by autoantigen-specific T cells. In the non-obese diabetic (NOD) mouse model, CD8+ T cells play an essential role in both the initial triggering of insulitis and its destructive phase, and proinsulin (PI) is one of the dominant target antigens (Ags). However, little is known about the beta cell epitopes presented by HLA class I molecules and recognized by human CD8+ T cells. We and other groups recently applied reverse immunology approaches to identify HLA class I-restricted PI epitopes. To establish an inventory of potential naturally processed epitopes, whole human PI or the transitional region between the B-chain and C-peptide were digested with purified proteasome complexes. By combining proteasome digestion data with epitope prediction algorithms, candidate epitopes restricted by HLA-A2.1 and other HLA class I molecules were identified. We validated immunogenicity and natural processing of the identified PI epitopes in HLA-A2.1-transgenic mice, while others demonstrated recognition of multiple PI epitopes by CD8+ T cells from T1DM and healthy subjects in the context of different HLA class I molecules. These results demonstrate the power of reverse immunology strategies for epitope discovery. DNA vaccination of HLA-transgenic mice may be another rapid and efficient reverse immunology approach to map additional epitopes derived from other T1DM Ags, such as IA-2 and glutamic acid decarboxylase 65 (GAD 65). Transfer of this information to Elispot- and MHC tetramer-based assay formats should allow to reliably detect and characterize autoreactive CD8+ T cell responses in T1DM, and may open new avenues for early T1DM diagnosis and immune intervention.
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PMID:HLA class I epitope discovery in type 1 diabetes. 1713 May 54


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