MHC class I antigen processing of an adenovirus CTL epitope is linked to the levels of immunoproteasomes in infected cells.

Proteasomes are the major source for the generation of peptides bound by MHC class I molecules. To study the functional relevance of the IFN-gamma-inducible proteasome subunits low molecular mass protein 2 (LMP2), LMP7, and mouse embryonal cell (MEC) ligand 1 in Ag processing and concomitantly that of immunoproteasomes, we established the tetracycline-regulated mouse cell line MEC217, allowing the titrable formation of immunoproteasomes. Infection of MEC217 cells with Adenovirus type 5 (Ad5) and analysis of Ag presentation with Ad5-specific CTL showed that cells containing immunoproteasomes processed the viral early 1B protein (E1B)-derived epitope E1B192-200 with increased efficiency, thus allowing a faster detection of viral entry in induced cells. Importantly, optimal CTL activation was already achieved at submaximal immunosubunit expression. In contrast, digestion of E1B-polypeptide with purified proteasomes in vitro yielded E1B192-200 at quantities that were proportional to the relative contents of immunosubunits. Our data provide evidence that the IFN-gamma-inducible proteasome subunits, when present at relatively low levels as at initial stages of infection, already increase the efficiency of antigenic peptide generation and thereby enhance MHC class I Ag processing in infected cells.

The proteasome inhibitor PI31 competes with PA28 for binding to 20S proteasomes.

PI31 is a previously described inhibitor of 20S proteasomes. Using recombinant PI31 we have analyzed its effect on proteasomal hydrolyzing activity of short fluorogenic substrates and of a synthetic 40-mer polypeptide. In addition, we investigated its influence on the activation of 20S proteasome by the proteasome activator PA28. PI31 inhibits polypeptide degradation already at concentrations which only partially inhibit fluorogenic substrate turnover and immunosubunits do not influence the PI31 binding affinity. Furthermore our data demonstrate that PI31 is a potent competitor of PA28-mediated activation.

Mutational analysis of subunit i beta2 (MECL-1) demonstrates conservation of cleavage specificity between yeast and mammalian proteasomes.

Proteasomes are the major protein-degrading complexes in the cytosol and regulate many cellular processes. To examine the functional importance of the MC14/MECL-1 proteasome active site subunits, cell lines expressing a catalytically inactive form of MECL-1 were established. Whereas mutant MECL-1 was readily incorporated into cytosolic proteasomes, replacing the constitutive MC14 subunit, removal of the prosequence was incomplete indicating that its processing required autocatalytic cleavage. Functional analyses showed that the absence of the MC14/MECL-1 active sites abrogated proteasomal trypsin-like activity, but did not affect other catalytic activities. Our data demonstrate a conservation of cleavage specificity between mammalian and yeast proteasomes.

The subunits MECL-1 and LMP2 are mutually required for incorporation into the 20S proteasome.

Processing of antigens for presentation by major histocompatibility complex (MHC) class I molecules requires the activity of the proteasome. The 20S proteasome complex is composed of 14 different subunits, 2 of which can be substituted by the interferon gamma (IFN-gamma)-inducible and MHC-encoded subunits LMP2 and LMP7 (low molecular mass poylpeptides 2 and 7). A third subunit, MECL-1, is inducible by IFN-gamma but is encoded outside the MHC. Here we show by cotransfection experiments that the incorporation of MECL-1 into the 20S proteasome is directly dependent on the expression of LMP2 but independent of LMP7. Conversely, the uptake of LMP2 is strongly enhanced by MECL-1 expression. The expression of MECL-1 caused a replacement of the homologous subunit Z in the 20S proteasome complex. LMP2 is required for MECL-1 incorporation at the level of proteasome precursor formation that guarantees the concerted incorporation of two IFN-gamma-inducible proteasome subunits encoded inside and outside the MHC. The obligatory coincorporation of MECL-1 and LMP2 is an important parameter for the interpretation of results obtained with LMP2-deficient cell lines and mice as well as for the design of experiments addressing the function of MECL-1 in antigen presentation.

Molecular cloning of the mouse proteasome subunits MC14 and MECL-1: reciprocally regulated tissue expression of interferon-gamma-modulated proteasome subunits.

The primary structures of the interferon-gamma-inducible mouse 20S proteasome subunit MECL-1 and its alternate homolog MC14 were determined. Northern analysis of mouse tissues revealed that MECL-1 mRNA predominantly occurred in thymus, lymph nodes, and spleen, whereas small amounts were detected in non-lymphoid tissues such as kidney, muscle, and testis. Unexpectedly, probing RNA blots with MC14 showed that tissues with high MECL-1 expression contained little MC14 and vice versa. A very similar reciprocal tissue expression was subsequently found for the homologous subunit pairs LMP2 and delta as well as LMP7 and MB1. The subunit protein composition of 20S proteasomes purified from liver, thymus, and lung reflected RNA expression. The impact of a regulated reciprocal tissue expression is discussed with respect to thymic selection and the induction of tolerance in potentially autoreactive T cells.

A third interferon-gamma-induced subunit exchange in the 20S proteasome.

The 20S proteasome is a protease complex of functional importance for antigen processing. Two of the 14 proteasome subunits, delta and MB1, can be replaced by the major histocompatibility complex (MHC)-encoded and interferon-gamma (IFN-gamma)-inducible subunits LMP2 and LMP7, respectively. LMP2 and LMP7 alter the cleavage site specificity of the 20S proteasome and are required for the efficient generation of T cell epitopes from a number of viral proteins and for optimal MHC class I cell surface expression. We compared the 20S proteasome subunit pattern from IFN-gamma-induced and non-induced mouse fibroblasts on two-dimensional gels and identified a third subunit exchange by microsequencing: the non-MHC-encoded subunit MECL-1 is induced by IFN-gamma and replaces a sofar barely characterized beta subunit designated 'MC14'. In analogy to LMP2 and LMP7, MECL-1 may be functional in MHC class I-restricted antigen presentation.

The interferon-gamma-inducible 11 S regulator (PA28) and the LMP2/LMP7 subunits govern the peptide production by the 20 S proteasome in vitro.

Antigenic peptides presented on major histocompatibility complex (MHC) class I molecules to cytotoxic T cells are generated in the cytosol by the 20 S proteasome. Upon stimulation of antigen presenting cells with interferon-gamma, two constitutive subunits of the 20 S proteasome are replaced by the MHC-encoded subunits low molecular mass polypeptide (LMP) 2 and LMP 7. In addition the expression of the two subunits of the 11 S regulator of the 20 S proteasome (PA28) are increased. As the function of LMP2 and LMP7 in antigen presentation is still controversial, we tested whether these subunits might operate by modifying proteasome activation through the 11 S regulator. We strongly overexpressed the two LMP subunits separately or together by transfection in murine fibroblasts. Isolated 20 S proteasomes from LMP transfectants were applied in digests of a 25-mer peptide in the presence or absence of a purified preparation of 11 S regulator from rabbit erythrocytes. Analysis of the cleavage products by high performance liquid chromatography and electrospray mass spectroscopy revealed marked differences in the peptide product profile in dependence on the LMP2 and LMP7 content. While the 11 S regulator did not preferentially activate LMP2 or 7 containing proteasomes, the binding of the 11 S regulator to any of the proteasome preparations markedly changed both the quality and quantity of peptides produced. These results suggest that the 11 S regulator increases the spectrum of peptides which can be generated in antigen presenting cells.

Molecular cloning and expression of subunit 12: a non-MCP and non-ATPase subunit of the 26 S protease.

A cDNA encoding subunit 12 (S12) of human erythrocyte 26 S protease has been isolated, sequenced and expressed. The cDNA contains an open reading frame that encodes a 36.6 kDA protein 96% identical to mouse Mov-34 and 67% identical to its Drosophila melanogaster homolog. Based on the high degree of sequence identity between human S12, mouse and Drosophila Mov-34 proteins, we conclude that the Mov-34 gene product is a component of the 26 S protease. Antibodies produced against two S12 fragments, Met1-Tyr95 (S12f95) and Met1-Leu205 (S12f205), react with S12 transferred to nitrocellulose from SDS-PAGE. In contrast, after transfer from native gels, the epitope(s) recognized by anti-S12f205 is exposed in the regulatory complex but appears to be masked when the regulatory complex associates with the multicatalytic protease.

Metabolism of phosphatidylinositides in rabbit red blood cells.

The particularly strong maturational loss of the monoester phosphate group turnover in PIP2 was demonstrated by in vivo as well as in vitro studies with rabbit reticulocytes. Both cycles of the phosphatidylinositide metabolism (PPI) were relatively insensitive against oxidative stress. Surprisingly, at high concentrations diamide did not alter the PIP2-, indeed, the PIP-turnover was drastically decreased.

Effect of diamide on polyphosphoinositide metabolism in red blood cells.

In intact red cells diamide had no strong influence on monoester phosphate group turnover of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid (PA) due to the potent GSH protection of these pathways in vivo. Following the complete GSH oxidation diamide impaired the turnover of PIP and PA dramatically. However that of PIP2 did not show any change. Indeed, a drastic irreversible decrease of PIP- and PA turnover was caused by a mild oxidative stress with diamide in G6PD deficient red cells.