Lothar Kuehn

Coordinated Dual Cleavages Induced by the Proteasome Regulator PA28 Lead to Dominant MHC Ligands

The eukaryotic 20S proteasome is known to associate with the IFN gamma-inducible regulator PA28. We analyzed the kinetics of product generation by 20S proteasomes with and without PA28. In the absence of PA28, the 20S proteasome rapidly generates peptides that have been cleaved only once, while internal fragments accumulate only slowly. In the presence of PA28, products generated by two flanking cleavages appear immediately as main products while the generation of single-cleavage products is strongly reduced. Kinetic data support a PA28-induced, coordinated double-cleavage mechanism. In particular, degradation of peptides derived from mouse cytomegalovirus pp89 and JAK1 kinase in the presence of PA28 leads to strongly enhanced production of the respective major histocompatibility complex ligands and potential precursors. These results show that PA28 profoundly alters the cleavage mechanism of the proteasome and appears to optimize the generation of dominant T-cell epitopes.

The multicatalytic proteinase (prosome) is ubiquitous from eukaryotes to archaebacteria

From the thermoacidophilic archaebacterium, Thermoplasma acidophilum, a proteolytically active particle has been isolated which is almost identical in size and shape with the multicatalytic proteinase (prosome) from rat. This result indicates that prosomes have been developed early in evolution and that they possibly serve functions common to all living cells.

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-γ, 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.

Electron microscopy and image analysis of the multicatalytic proteinase

On electron micrographs, negatively stained multicatalytic proteinase molecules are viewed end‐on (ring shaped) or side‐on (rectangular shaped). For aurothioglucose, ammonium molybdate‐ and phosphotungstate‐stained molecules, the dimensions measured are consistent. In contrast, uranyl acetate‐staining reveals ring‐shaped particles which vary in diameter between 12 and 16 nm. This is due to a partial collapse and substantial flattening of the structure. Digital image analysis of side‐on views of the particles reveals a tripartite, reel‐shaped structure. Within the ring‐like, end‐on projections of ammonium molybdate‐stained molecules six local centres of mass can be discerned; their position appears to depart, however, from a true six‐fold symmetry.

Drosophila small cytoplasmic 19S ribonucleoprotein is homologous to the rat multicatalytic proteinase

All eukaryotic cells so far analysed contain 19S particles which share a cylinder-like shape and are composed of a set of proteins of relative molecular mass ranging typically from 19,000 to 36,000 (refs 1–10). Proposed functions have included synthetase activity11, transfer RNA processing12 or messenger RNA repression6, but their biological importance remains obscure. A multicatalytic proteinase (MCP) of similar size and shape has been isolated from mammalian tissues13–24. The apparent similarities of these high molecular weight complexes suggest a biochemical and functional homology between the small cytoplasmic 19S particle from Drosophila melanogaster (19S-scRNP) (ref. 7) and rat MCP (ref. 14). By means of electron microscopy, immunological techniques, RNA identification and proteinase activity assays, we were able to show that the two structurally similar complexes are immunologically related ribonucleoproteins (RNPs) with similar proteolytic activity.

Subtypes of 20S proteasomes from skeletal muscle

20S proteasomes from tissues and cells are a mixture of several subtypes. From rat skeletal muscle we have tentatively separated six different subtypes of 20S proteasomes purified from rat skeletal muscle by high-resolution anion exchange chromatography. Immunoblot analysis using antibodies to the beta-subunits LMP2, LMP7 and their constitutive counterparts delta and MB1 revealed that two of the three major subtypes (subtypes I and II) are constitutive proteasomes, whereas two of the three minor subtypes belong to the subpopulation of immuno-proteasomes. Subtype III and IV are intermediate-type proteasomes. Enzymological characterisation of the six subtypes revealed clearly different V(max) values for hydrolysis of fluorogenic peptide substrates as well as significantly different activities measured with a 25-mer polypeptide of the murine cytomegalovirus IE pp89 protein as substrate. Our data show that the properties of 20S proteasomes isolated from a given tissue or cells are always the average of the properties of the whole set of proteasome subtypes.

Pronounced up‐regulation of the PA28α/β proteasome regulator but little increase in the steady‐state content of immunoproteasome during dendritic cell maturation

Dendritic cells (DC) are professional antigen‐presenting cells that activate CTL by presenting MHC class I‐restricted peptides that are processed through the proteasome pathway. Previously, wereported that upon DC maturation the synthesis is switched towards the exclusive production of immunoproteasomes containing the active site subunits LMP2, LMP7 and MECL‐1. In this study we investigated the mechanism by which proteasome assembly is regulated in mature DC. Quantitative analysis of mRNA expression showed very limited transcriptional induction of LMP7, MECL‐1 and UMP1 in mature DC and a moderate mRNA increment for LMP2 and PA28α and β. We investigated a role of PA28α/β in regulating proteasome assembly in DC. PA28α/β coprecipitated with 13S/16S proteasome precursor complexes but associated with mature constitutive and immunoproteasomes to the same extent. Furthermore, we determined the steady‐state proteasome subunit composition in DC. Replacement of constitutive proteasomes by immunoproteasomes in maturing DC was very slow and occurred only to a minor extent. Our data suggest that the limited turnover of 20S proteasomes in mature DC probably contributes little to recently reported marked differences in antigen presentation between immature and mature DC and that alternative mechanisms may be responsible for this phenomenon.

Size and shape of the multicatalytic proteinase from rat skeletal muscle

The multicatalytic proteinase from rat skeletal muscle, a non-lysosomal high molecular weight enzyme active at neutral to alkaline pH, has been examined in the electron microscope as well as by dynamic laser light scattering. Both methods reveal monodisperse particles. Electron micrographs show a cylinder-shaped complex with a diameter of 11 nm and a length of 16 nm in negatively stained, and a diameter of 9.6 nm and a length of 14.3 nm in freeze-dried, heavy metal replicated specimens. The molecule is composed of four rings or disks.

Identification of three high molecular mass cysteine proteinases from rat skeletal muscle.

Three cysteine proteinases were isolated from the post‐myofibrillar fraction of rat skeletal muscle. Proteinase I preferentially hydrolyzes Z‐Phe—Arg‐NMec with pH optimum at 8–9. The enzyme activity is stabilized by ATP against thermal inactivation. Proteinase II and III were not resolved by anion‐exchange chromatography, by affinity chromatography on Arginine—Sepharose or by gel filtration. Proteinase II, splitting Bz‐Val‐Gly‐Arg‐NMec optimally at pH 10–10.5, is inactivated by ATP, whereas Proteinase III, hydrolyzing Suc‐Ala‐Ala‐Phe‐NMec at pH 7–7.5 is not affected by the nucleotide. The molecular mass of proteinase I is about 750 000 and that of proteinase II and III is about 650 000, as determined by gel filtration.

Reconstitution of proteasome activator PA28 from isolated subunits: optimal activity is associated with an α,β-heteromultimer

PA28, a 200 kDa activator of 20S proteasomes, was purified from human placenta and was gel electrophoretically resolved into two different subunits, α and β. In reconstitution experiments, α‐subunits alone were found to re‐associate forming homooligomers with an M r of about 200 kDa, which elicit a stimulatory effect on proteasomal peptide‐hydrolyzing activity, albeit at a moderate level. Under the same conditions, isolated β‐subunits were neither found to associate nor did they display stimulatory activity. Significantly, when both α‐ and β‐subunits were present in the reconstitution assay, heteromultimers formed, concomitant with a marked increase in stimulatory activity when compared with that of α‐homooligomers. The reconstituted PA28α,β protein is indistinguishable from purified PA28 by several criteria: it displays the same molecular mass, shows the same abundance of α‐ and β‐subunits and has a similar stimulatory activity toward 20S proteasomes. These results indicate that optimal PA28 activity is associated with a heteromultimeric structure which contains the α‐ and β‐subunits in fixed stoichiometry, most likely as an α 3 β 3‐heterohexamer.