Role of immunoproteasomes and thymoproteasomes in health and disease

The proteasome is a multisubunit protease that degrades intracellular proteins into small peptides. Besides playing a pivotal role in many cellular processes indispensable for survival, it is involved in the production of peptides presented by major histocompatibility complex class I molecules. In addition to the standard proteasome shared in all eukaryotes, jawed vertebrates have two specialized forms of proteasome known as immunoproteasomes and thymoproteasomes. The immunoproteasome, which contains cytokine-inducible catalytic subunits with distinct cleavage specificities, produces peptides presented by class I molecules more efficiently than the standard proteasome. The thymoproteasome, which contains a unique catalytic subunit β5t, is a tissue-specific proteasome expressed exclusively in cortical thymic epithelial cells. It plays a critical role in CD8⁺ cytotoxic T cell development via positive selection. This review provides a brief overview on the structure and function of these specialized forms of proteasome and their involvement in human disease.     

Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules

Most antigenic peptides presented by MHC class I molecules result from the degradation of intracellular proteins by the proteasome. In lymphoid tissues and cells exposed to IFNγ, the standard proteasome is replaced by the immunoproteasome, in which all of the standard catalytic subunits β1, β2, and β5 are replaced by their inducible counterparts β1i, β2i, and β5i, which have different cleavage specificities. The immunoproteasome thereby shapes the repertoire of antigenic peptides. The existence of additional forms of proteasomes bearing a mixed assortment of standard and inducible catalytic subunits has been suggested. Using a new set of unique subunit-specific antibodies, we have now isolated, quantified, and characterized human proteasomes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (β5i) or two (β1i and β5i) of the three inducible catalytic subunits of the immunoproteasome. These intermediate proteasomes represent between one-third and one-half of the proteasome content of human liver, colon, small intestine, and kidney. They are also present in human tumor cells and dendritic cells. We identified two tumor antigens of clinical interest that are processed exclusively either by intermediate proteasomes β5i (MAGE-A3(271-279)) or by intermediate proteasomes β1i-β5i (MAGE-A10(254-262)). The existence of these intermediate proteasomes broadens the repertoire of antigens presented to CD8 T cells and implies that the antigens presented by a given cell depend on their proteasome content.     

Low-molecular-weight protein (LMP)2/LMP7 abnormality underlies the downregulation of human leukocyte antigen class I antigen in a hepatocellular carcinoma cell line

BACKGROUND: Tumor cells may alter the expression of numerous components involved in antigen-processing machinery to decrease human leukocyte antigen (HLA) class I expression, allowing the tumor cells to escape immune surveillance. The purpose of the present study was to investigate the involvement of these components in the downregulation of HLA class I expression in human hepatocellular carcinoma cell line BEL7,404. METHODS: Expression of HLA-I and antigen presentation-related genes were analyzed by flow cytometry and polymerase chain reaction. The HLA class I-deficient BEL7,404 cell was transfected with the low-molecular-weight protein (LMP) 2 and LMP7 gene and were analyzed by flow cytometry for restoration of surface HLA class I expression. RESULTS: The BEL7,404 cells downregulated the expression of HLA class I antigen and lacked expression of LMP2 and LMP7. Interferon (IFN)-gamma treatment increased the expression of LMP2 but not LMP7. The LMP2-transfected BEL7,404 cells or LMP2 and LMP7-cotransfected cells restored surface HLA class I expression while LMP7-transfected cells did not. However, in IFN-gamma-treated BEL7,404 cells, transfection with the LMP7 gene induced more HLA class I expression than mock transfection. CONCLUSIONS: The LMP2 gene was required for the expression of HLA class I molecules in BEL7,404. The LMP7 was not the major reason for loss of HLA class I in BEL7,404 cells, although the supply of exogenous LMP7 could increase surface expression of HLA class I antigen.     

Prophylactic and therapeutic efficacies of a selective inhibitor of the immunoproteasome for Hashimoto's thyroiditis, but not for Graves' hyperthyroidism, in mice

Major histocompatibility complex (MHC) class I-restricted T cell epitopes are generated mainly by the immunoproteasome in antigen-presenting cells. Therefore, inhibition of activity of this proteolytic complex molecule is thought to be a potential treatment for cell-mediated autoimmune diseases. We therefore studied the efficacy of an immunoproteasome inhibitor, ONX 0914 (formerly PR-957), for the treatment of autoimmune thyroid diseases, including cell-mediated Hashimoto's thyroiditis and autoantibody-mediated Graves' hyperthyroidism using mouse models. Our data show that ONX 0914 was effective prophylactically and therapeutically at suppressing the degree of intrathyroidal lymphocyte infiltration and, to a lesser degree, the titres of anti-thyroglobulin autoantibodies in non-obese diabetic (NOD)-H2(h4) mice, an iodine-induced autoimmune thyroiditis model. It also inhibited differentiation of T cells to T helper type 1 (Th1) and Th17 cells, effector T cell subsets critical for development of thyroiditis in this mouse strain. In contrast, its effect on the Graves' model was negligible. Although ONX 0914 exerts its immune-suppressive effect through not only suppression of immune proteasome but also other mechanism(s), such as inhibition of T cell differentiation, the present results suggest that the immunoproteasome is a novel drug target in treatment of Hashimoto's thyroiditis in particular and cell-mediated autoimmune diseases in general.     

免疫蛋白酶体亚单位低分子量多肽7(β5i)的研究进展

泛素-蛋白酶体系统(UPS)是真核细胞内非溶酶体通路的蛋白质降解主要途径,UPS水解活性与β1、β2、β5亚基密切相关,在特定的条件下,β1、β2、β5亚基可被相应的免疫亚基β1i、β2i和低分子量多肽7(LMP7或β5i)替代,其中β5i活性最强,新形成的结构被称为免疫蛋白酶体(immunoproteasome)。β5i在自身免疫性疾病、心脑血管疾病、肥胖与代谢疾病、肿瘤等方面发挥着重要作用。本文综述了β5i在上述疾病中的研究进展,以便为药物治疗提供理论依据,从而改善疾病预后。     

A MAGE-C2 antigenic peptide processed by the immunoproteasome is recognized by cytolytic T cells isolated from a melanoma patient after successful immunotherapy

We have pursued our analysis of a melanoma patient who showed almost complete tumor regression following vaccination with MAGE-A1 and MAGE-A3 antigens. We previously described high frequencies of tumor-specific CTL precursors in blood samples collected after but also before vaccination. A set of CTL clones were derived that recognized antigens different from those of the vaccine. Two of these antigens were peptides encoded by another MAGE gene, MAGE-C2. Here we describe the antigen recognized by another tumor-specific CTL clone. It proved to be a third antigenic peptide encoded by gene MAGE-C2, ASSTLYLVF. It is presented by HLA-B57 molecules and proteasome-dependent. Tumor cells exposed to interferon-gamma (IFN-γ) were better recognized by the anti-MAGE-C2(42-50) CTL clone. This mainly resulted from a better processing of the peptide by the immunoproteasome as compared to the standard proteasome. Mass spectrometric analyses showed that the latter destroyed the antigenic peptide by cleaving between two internal hydrophobic residues. Despite its higher "chymotryptic-like" (posthydrophobic) activity, the immunoproteasome did not cleave at this position, in line with the suggestion that hydrophobic residues immediately downstream from a cleavage site impair cleavage by the immunoproteasome. We previously reported that one of the other MAGE-C2 peptides recognized by CTL from this patient was also better processed by the immunoproteasome. Together, these results support the notion that the tumor regression of this patient was mediated by an antitumor response shaped by IFN-γ and dominated by CTL directed against peptides that are better produced by the immunoproteasome, such as the MAGE-C2 peptides.     

Immunoproteasome deficiency is a feature of non-small cell lung cancer with a mesenchymal phenotype and is associated with a poor outcome

The immunoproteasome plays a key role in generation of HLA peptides for T cell-mediated immunity. Integrative genomic and proteomic analysis of non-small cell lung carcinoma (NSCLC) cell lines revealed significantly reduced expression of immunoproteasome components and their regulators associated with epithelial to mesenchymal transition. Low expression of immunoproteasome subunits in early stage NSCLC patients was associated with recurrence and metastasis. Depleted repertoire of HLA class I-bound peptides in mesenchymal cells deficient in immunoproteasome components was restored with either IFNγ or 5-aza-2′-deoxycytidine (5-aza-dC) treatment. Our findings point to a mechanism of immune evasion of cells with a mesenchymal phenotype and suggest a strategy to overcome immune evasion through induction of the immunoproteasome to increase the cellular repertoire of HLA class I-bound peptides.Proteasomes are multisubunit complexes that degrade intracellular proteins through the ubiquitin–proteasome pathway (1). The three catalytic β subunits β1, β2, and β5 in the proteasome complex are replaced by proteasome (prosome, macropain) subunit B9 (PSMB9)/β1i, PSMB10/β2i, and PSMB8/β5i, respectively, to form the immunoproteasome (2). The immunoproteasome generates peptides suitable for binding onto HLA I molecules, facilitating antigen presentation for CD8⁺ T-cell responses. Lack of expression or down-regulation of the immunoproteasome may contribute to immune evasion through antigen loss (3).The impact of immunoproteasome expression on antigen presentation in tumors of epithelial origin is not well established. We have investigated the constitutive and induced expression patterns of immunoproteasome subunits in non-small cell lung carcinoma (NSCLC) and their impact on antigen presentation. We provide evidence for dysregulated expression of immunoproteasome subunits in NSCLC cells with a mesenchymal phenotype, associated with a markedly reduced repertoire of HLA-bound peptides. Reduced expression of immunoproteasome subunits in NSCLC was also associated with reduced disease free survival.     

Argyrin B,a non‐competitive inhibitor of the human immunoproteasome exhibiting preference for β1i

Inhibitors of the proteasome have found broad therapeutic applications; however, they show severe toxicity due to the abundance of proteasomes in healthy cells. In contrast, inhibitors of the immunoproteasome, which is upregulated during disease states, are less toxic and have increased therapeutic potential including against autoimmune disorders. In this project, we report argyrin B, a natural product cyclic peptide to be a reversible, non‐competitive inhibitor of the immunoproteasome. Argyrin B showed selective inhibition of the β5i and β1i sites of the immunoproteasome over the β5c and β1c sites of the constitutive proteasome with nearly 20‐fold selective inhibition of β1i over the homologous β1c. Molecular modelling attributes the β1i over β1c selectivity to the small hydrophobic S1 pocket of β1i and β5i over β5c to site‐specific amino acid variations that enable additional bonding interactions and stabilization of the binding conformation. These findings facilitate the design of immunoproteasome selective and reversible inhibitors that may have a greater therapeutic potential and lower toxicity.