La regla de Taylor para la tasa de interés

al., 1993). The ubiquitin system is a non-lysosomal ATP-dependent proteolytic pathway in which the covalent attachment of ubiquitin, a highly conserved 76- residue polypeptide, is thought to mark proteins destined for degradation (Hershko and Ciechanouer, 1992). Successive enzyme action leads to the formation of isopeptide bonds between the e-amino groups of Lys residues in the substrate protein and the carboxyl terminus of ubiquitin. Ubiquitin is then itself ubiquitinated. The reaction is repeated, resulting in the formation of large conjugates that are specifically recognised by the 26S proteasome and degraded.

6.1.2. Involvement of the proteasome in the class I antigen processing pathway

The involvement of the proteasome in antigen processing was first suggested in 1989 (Townsend and Bodmer, 1989). A pathway was proposed suggesting that antigenic proteins were degraded, in the cytoplasm, into peptides which were transported through the ER. These peptides then bound to MHC class I molecules and travelled to the cell surface for presentation to T cells. It was proposed that the proteasome was involved in such peptide production and recently evidence has been produced to support this hypothesis.

A group of 16 proteins of molecular weight 15-30kD were precipitated from mouse cell lines using anti-MHC alloantisera and were called Low Molecular W eight Proteins, or LMPs (Monaco and McDevitt, 1982). The LMPs were expressed on monocyte and macrophage cell lines, as well as normal macrophages and spleen cells. Precipitation of LMPs from mice containing cross-overs within the H-2 region with an anti-H-2^ serum led to the discovery that two subunits responsible for such precipitation were genetically linked to the murine class II region. These two subunits, named LMP2 and LMP7, were localised to a region of the mouse MHC between the genes Oa and Ob, the equivalents of the DNA and

It was pointed out that the 20S proteasome resembled the LMPs in size and subunit composition (Parham, 1990) and a relationship between the two com plexes was soon discovered. A nti-L M P and an ti-p ro teaso m e immunoprecipitates were compared from a murine macrophage cell extract (Brown et al., 1991). An anti-H-2‘* antiserum was used to immunoprecipitate LMP antigens and a rabbit antiserum was made against purified rat liver proteasomes. Mixing of LMP and proteasome precipitates before electrophoresis demonstrated that 16 LMP subunits migrated with identical mobility to 16 of the polypeptides in the anti-proteasome precipitate. Two proteasome subunits migrated identically to the MHC-linked LMP2 and LMP7 polypeptides indicating that normal proteasomes contained these subunits. However, four proteasome subunits were not found in LMP precipitates and proteasome could still be precipitated from lysate that had been precleared with anti H-2^ serum. This suggests that although the LMP and proteasome complexes are similar they are not identical. The precise relationship between the LMP complex and the proteasome remains to be elucidated.

6.1.3. LM P2 and LMP7: two proteasome subunits localised w ithin the M HC class II region

Screening of cDNA libraries with cosmid clones spanning the MHC class II region DNA to DOB in the human and Pb to Ob in the mouse allowed the precise localisation of the two genes LMP2 and LMP7 (Glynne et al., 1991; Martinez and Monaco, 1991; Kelly et al., 1991). In the human, these genes flanked the TAPI

transporter gene with the order being LMP2, TAPI, LMP7 and TAP2. Sequence comparisons confirmed that these two genes were members of the proteasome complex. The localisation of LMP2 and LMP 7 within the MHC and their close proximity to the transporter genes strongly implicates the proteasome as having a role in antigen processing. The immune system, during the course of evolution, may have recruited a previously existing structure required for normal cellular turnover by attaching two extra subunits that serve to direct its output to the TAP

transporter. Alternatively, the two MHC-linked subunits may serve to modify the proteolytic action of the proteasome to produce peptides better suited for MHC class I binding. Recently, several reports have provided data supporting the latter hypothesis (Gaczynska et al., 1993; Gaczynska et al., 1994; Rock et al., 1994).

As mentioned above, the proteasome exhibits multiple peptidase activities including three activities that preferentially hydrolyse small peptides on the carboxyl side of hydrophobic, basic or acidic residues. These activities are regulated by the cytokine interferon y (IFN-y) which is a stimulator of MHC class I presentation. Treatment of cells with IFN-y increases the capacity of the proteasomes to cleave oligopeptides after hydrophobic and basic residues and reduces cleavage after acidic residues (Gaczynska et al., 1993). IFN-y treatment also induces the addition of the LMP2 and LMP7 subunits to the proteasome and the loss of two other units (Yang et al., 1992). These changes in peptidase activity are not seen on IFN-y treatment of the deletion mutant 721.174 which lacks the

LMP2 and L M P 7 genes. These findings led to the suggestion that the incorporation of LMP2 and -7 into the proteasome was responsible for the alterations in peptidase activities.

This hypothesis was investigated in the following manner. LMP2 and LMP7

cDNAs were either transfected into HeLa cells or 721.174 B-LCLs (Gaczynska et al., 1994). Proteasomes were then isolated from transfectant and control cells, the levels of the LMP2 and LMP 7 subunits assessed, and their peptidase activities compared. LMP7 transfection into the strain 721.174 enhanced the hydrophobic and basic site activities, whilst LMP2 transfection lowered the acidic site activity. Similarly, transfection of HeLa cells with LMP2 and LMP7 mimicked the effects of IFN-y treatment, which enhances hydrophobic and basic activities and reduces cleavage after acidic residues. This effect favours the generation of the kinds of oligopeptides that preferentially bind to MHC class I molecules providing circumstantial evidence for involvement of the proteasome in antigen processing.

The final piece of evidence confirming the role of the proteasome in the class I processing pathway appeared recently (Rock et al., 1994). Ovalbumin was introduced into the cytosol of murine B lymphoblastoid cells and the effect of peptide aldehydes, which are known to inhibit peptidase activities, on its presentation was tested. When antigen presenting cells were exposed to the peptide aldehyde, presentation of ovalbumin was inhibited in a dose dependent manner up to 100%. Ovalbumin presentation required the TAP transporter and was inhibited by brefeldin A. Aldehydes had no effect on the presentation of ovalbumin-derived peptide in cells infected with a vaccinia viral construct containing a minigene encoding this peptide. Therefore, aldehydes selectively blocked the production of the immunogenic peptide and not any subsequent step in the class I pathway. All these results show that the proteasome is responsible

for the generation of peptides presented to MHC class I molecules and thus has a major role in the class I antigen processing pathway.