Pertussis toxin B-pentamer mediates intercellular transfer of membrane proteins and lipids.

Pertussis toxin (PTx) is the major virulence factor of Bordetella pertussis. The enzymatic or active (A) subunit inactivates host G protein coupled receptor (GPCR) signaling pathways. The non-enzymatic binding (B) subunit also mediates biological effects due to lectin-like binding characteristics, including the induction of T cell receptor (TCR) signaling and subsequent down-regulation of chemokine receptor expression. Here we report another activity attributable to PTxB, facilitating transfer of membrane material between mammalian cells. This activity does not require the TCR, and does not require cell-to-cell contact or cellular aggregation. Rather, membrane vesicles are transferred from donor to recipient cells in a toxin-dependent fashion. Membrane transfer occurs in different cell types, including cultured human T cells, CHO cells, and human primary peripheral blood mononuclear cells. Transfer involves both lipid and integral membrane proteins, as evidenced by the transfer of T and B cell-specific receptor molecules to other PBMCs. Interestingly, membrane transfer activity is a property that PTx shares with some, but not all, cell-aggregating lectins that are mitogenic for human T cells, and appears to be related to the ability to bind certain host cell glycolipids. This phenomenon may represent another mechanism by which pertussis toxin disrupts mammalian intra- and inter-cellular signaling.

A critical role for the inducible proteasomal subunits LMP7 and MECL1 in cytokine production by activated murine splenocytes.

BACKGROUND AND PURPOSE: The proteasome is a multi-subunit complex that proteolytically cleaves proteins. The replacement of the constitutive proteasome subunits ß1, ß2, and/or ß5 with the IFNγ-inducible subunits LMP2, MECL1, and/or LMP7 results in the 'immunoproteasome'. The inducible subunits change the cleavage specificities of the proteasome, but it is unclear whether they have functions in addition to this. The purpose of the present study was to determine the role of the proteasome in general, as well as LMP7 and MECL1 specifically, with regard to cytokine production by activated primary splenocytes. METHODS: A LMP7/MECL1-null mouse was engineered to determine the roles of these subunits in cytokine production. Isolated splenocytes from wild-type and LMP7/MECL1-/- mice were treated with lactacystin and activated with PMA and ionomycin and subsequently cytokine mRNA levels were quantified. RESULTS: The present study demonstrates that LMP7/MECL1 regulates the expression of IFNγ, IL4, IL10, IL2Rß, GATA3, and t-bet. In contrast, the regulation of IL2, IL13, TNFα, and IL2Rα by the proteasome appears to occur independently of LMP7/MECL1. CONCLUSIONS: Collectively, the present study demonstrates that LMP7 and MECL1 regulate cytokine expression, suggesting this system represents a novel mechanism for the regulation of cytokines and cytokine signaling.

The immunoproteasomes regulate LPS-induced TRIF/TRAM signaling pathway in murine macrophages.

We have proposed the novel concept that the macrophage ubiquitin-proteasome pathway functions as a key regulator of Lipopolysaccharide (LPS)-induced inflammation signaling. These findings suggest that proteasome-associated protease subunits X, Y, and Z are replaced by LMP subunits after LPS treatment of RAW 264.7 cells. The objective here was to determine the contribution of selective LMP proteasomal subunits to LPS-induced nitric oxide (NO) and TNF-α production in primary murine macrophages. Accordingly, thioglycollate-elicited macrophages from LMP7, LMP2, LMP10 (MECL-1), and LMP7/MECL-1 double knockout mice were stimulated in vitro with LPS, and were found to generate markedly reduced NO levels compared to wild-type (WT) mice, whereas TNF-α levels responses were essentially unaltered relative to wild-type responses. The recent studies suggest that the TRIF/TRAM pathway is defective in LMP knockouts which may explain why iNOS/NO are not robustly induced in LPS-treated macrophages from knockouts. Treating these macrophages with IFN-γ and LPS, however, reverses this defect, leading to robust NO induction. TNF-α is induced by LPS in the LMP knockout macrophages because IκB and IRAK are degraded normally via the MyD88 pathway. Collectively, these findings strongly support the concept that LMP7/MECL-1 proteasomes subunits actively function to regulate LPS-induced NO production by affecting the TRIF/TRAM pathway.

Rates of processing determine the immunogenicity of immunoproteasome-generated epitopes.

CD8 T cells resolve intracellular pathogens by responding to pathogen-derived peptides that are presented on the cell surface by MHC class I molecules. Although most pathogens encode a large variety of antigenic peptides, protective CD8 T cell responses target usually only a few of these. To determine the mechanism by which the IFN-gamma-inducible proteasome (immuno) subunits enhance the ability of specific pathogen-derived peptides to elicit CD8 T cell responses, we generated a recombinant Listeria monocytogenes strain (rLM-E1) that secretes a model Ag encompassing the immunoproteasome-dependent E1B(192-200) and immunoproteasome-independent E1A(234-243) epitope. Analyses of Ag presentation showed that infected gene-deficient professional APCs, lacking the immunosubunits LMP7/ibeta5 and MECL-1/ibeta2, processed and presented the rLM-E1-derived E1B(192-200) epitope but with delayed kinetics. E1A epitope processing proceeded normally in these cells. Accordingly, infected gene-deficient mice failed to respond to the otherwise immunodominant E1B(192-200) epitope but mounted normal CD8 T cell responses to E1A(234-243) which was processed by the same professional APCs, from the same rLM-E1 Ag. The inability of gene-deficient mice to respond to E1B(192-200) was not explained by insufficient quantities of antigenic peptide, as splenic APC of 36-h-infected gene-deficient mice that presented the two E1 epitopes at steady state levels elicited responses to both E1B(192-200) and E1A(234-243) when transferred into LMP7+MECL-1-deficient mice. Taken together, our findings indicate that not absolute epitope quantities but early Ag-processing kinetics determine the ability of pathogen-derived peptides to elicit CD8 T cell responses, which is of importance for rational T cell vaccine design.

Ubiquitin- and ATP-independent proteolytic turnover of p21 by the REGgamma-proteasome pathway.

We previously demonstrated that the proteasome activator REGgamma directs degradation of the steroid receptor coactivator SRC-3 by the 20S proteasome in an ATP- and ubiquitin-independent manner. Our efforts to identify additional endogenous direct targets of the REGgamma proteasome revealed that p21(Waf/Cip1), a central cyclin-dependent kinase inhibitor, is another endogenous target. Gain-of-function analysis, RNAi knockdown, REGgamma-deficient MEF analysis, and pulse-chase experiments substantiate that REGgamma promotes degradation of unbound p21. Cell-free proteasome proteolysis assays using purified REGgamma, p21, and the 20S proteasome confirm that REGgamma directly mediates degradation of free p21 in an ATP- and ubiquitin-independent manner. Depletion of REGgamma in a thyroid carcinoma cell line results in cell-cycle and proliferative alterations. Our study reveals that, in addition to degrading the SRC-3 growth coactivator, REGgamma also has a role in the regulation of the cell cycle through its ability to influence the level of a cell-cycle regulator(s).

Immunoproteasome subunit deficiencies impact differentially on two immunodominant influenza virus-specific CD8+ T cell responses.

Primary CD8+ T cell (T(CD8+)) responses to viruses are directed toward multiple Ags and shaped by both the level of Ag presentation and the underlying Ag-specific T(CD8+) repertoire. The relative importance of these factors in deciding the hierarchy of T(CD8+) responses and how they are influenced by the immunoproteasome are not well understood. Using an influenza infection model in mice deficient in various immunoproteasome subunits, we observe that Ag presentation and T(CD8+) repertoire are altered in an epitope-specific and immunoproteasome subunit-dependent manner. More importantly, we find that the level of Ag presentation and the extent of the underlying repertoire can work either alone or in concert to determine definitively the magnitude of the individual T(CD8+) responses and hence the overall T(CD8+) hierarchy. Together, these results provide a clearer understanding of how immunodominance hierarchies are established.

An altered T cell repertoire in MECL-1-deficient mice.

Immunoproteasome subunits low-molecular mass polypeptide (LMP)2 and LMP7 affect Ag presentation by MHC class I molecules. In the present study, we investigated the function of the third immunosubunit LMP10/multicatalytic endopeptidase complex-like (MECL)-1 (beta2i) in MECL-1 gene-targeted mice. The number of CD8+ splenocytes in MECL-1-/- mice was 20% lower than in wild-type mice. Infection with lymphocytic choriomeningitis virus (LCMV) elicited a markedly reduced cytotoxic T cell (CTL) response to the LCMV epitopes GP276-286/Db and NP205-212/Kb in MECL-1-/- mice. The weak CTL response to GP276-286/Db was not due to an impaired generation of this epitope but was attributed to a decreased precursor frequency of GP276-286/Db-specific T cells. The expansion of TCR-Vbeta10+ T cells, which contain GP276-286/Db-specific cells, was reduced in LCMV-infected MECL-1-/- mice. Taken together, our data reveal an in vivo function of MECL-1 in codetermining the T cell repertoire for an antiviral CTL response.

T cells lacking immunoproteasome subunits MECL-1 and LMP7 hyperproliferate in response to polyclonal mitogens.

Immunoproteasomes comprise a specialized subset of proteasomes that is defined by the presence of three catalytic immunosubunits: LMP2, MECL-1 (LMP10), and LMP7. Proteasomes in general serve many cellular functions through protein degradation, whereas the specific function of immunoproteasomes has been thought to be largely, if not exclusively, optimization of MHC class I Ag processing. In this report, we demonstrate that T cells from double knockout mice lacking two of the immunosubunits, MECL-1 and LMP7, hyperproliferate in vitro in response to various polyclonal mitogens. We observe hyperproliferation of both CD4(+) and CD8(+) T cell subsets and demonstrate accelerated cell cycling. We do not observe hyperproliferation of T cells lacking only one of these subunits, and thus hyperproliferation is independent of either reduced MHC class I expression in LMP7(-/-) mice or reduced CD8(+) T cell numbers in MECL-1(-/-) mice. We observe both of these latter two phenotypes in MECL-1/LMP7(-/-) mice, which indicates that they also are independent of each other. Finally, we provide evidence of in vivo T cell dysfunction by demonstrating increased numbers of central memory phenotype CD8(+) T cells in MECL-1/LMP7(-/-) mice. In summary, this novel phenotype of hyperproliferation of T cells lacking both MECL-1 and LMP7 implicates a specific role for immunoproteasomes in T cell proliferation that is not obviously connected to MHC class I Ag processing.

Immune defects in 28-kDa proteasome activator gamma-deficient mice.

Protein complexes of the 28-kDa proteasome activator (PA28) family activate the proteasome and may alter proteasome cleavage specificity. Initial investigations have demonstrated a role for the IFN-gamma-inducible PA28alpha/beta complex in Ag processing. Although the noninducible and predominantly nuclear PA28gamma complex has been implicated in affecting proteasome-dependent signaling pathways, such as control of the mitotic cell cycle, there is no previous evidence demonstrating a role for this structure in Ag processing. We therefore generated PA28gamma-deficient mice and investigated their immune function. PA28gamma(-/-) mice display a slight reduction in CD8+ T cell numbers and do not effectively clear a pulmonary fungal infection. However, T cell responses in two viral infection models appear normal in both magnitude and the hierarchy of antigenic epitopes recognized. We conclude that PA28gamma(-/-) mice, like PA28alpha(-/-)/beta(-/-) mice, are deficient in the processing of only specific Ags.

Association of beta2-microglobulin with the alpha3 domain of H-2Db heavy chain.

MHC class I molecules are heterotrimeric complexes composed of heavy chain, beta2-microglobulin (beta2m) and short peptide. This trimeric complex is generated in the endoplasmic reticulum (ER), where a peptide loading complex (PLC) facilitates transport from the cytosol and binding of the peptide to the preassembled ER resident heavy chain/beta2m dimers. Association of mouse MHC class I heavy chain with beta2m is characterized by allelic differences in the number and/or positions of amino acid interactions. It is unclear, however, whether all alleles follow common binding patterns with minimal contributions by allele-specific contacts, or whether essential contacts with beta2m are different for each allele. While searching for the PLC binding site in the alpha3 domain of the mouse MHC class I molecule H-2Db, we unexpectedly discovered a site critical for binding mouse, but not human, beta2m. Interestingly, amino acids in the corresponding region of another MHC class I heavy chain allele do not make contacts with the mouse beta2m. Thus, there are allelic differences in the modes of binding of beta2m to the heavy chain of MHC class I.

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.

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.