Sequence information within proteasomal prosequences mediates efficient integration of beta-subunits into the 20 S proteasome complex.

The maturation of proteases is governed by prosequences. During the biogenesis of the highly oligomeric eukaryotic 20 S proteasome five different prosequence-containing subunits have to be integrated and processed either by autocatalysis or by neighbouring subunits. To analyse the functional impact of proteasomal prosequences during complex formation, the propeptide of the facultative subunit beta1i/LMP2 was truncated to nine amino acid residues or completely deleted. Additionally, the charged residues within the truncated beta1i/LMP2 version were replaced by neutral residues. While deletion did not affect subunit incorporation, the presence of charged residues within the truncated version of the LMP2 propeptide diminished incorporation efficiency, an effect that was restored upon replacement of the charged amino acids against neutral components. During immunoproteasome formation, incorporation and processing of inducible proteasome beta-subunits are cooperative processes. We demonstrate a linear correlation of the levels of beta2i/MECL1 and beta1i/LMP2 within 20 S proteasomes, suggesting a physical interaction to be the molecular basis for the biased incorporation of both subunits. In the absence of beta5i/LMP7, precursor complexes containing unprocessed beta1i/LMP2 accumulated. The contribution of beta5i/LMP7 on the cooperative formation of a homogeneous population of immunoproteasome is therefore most likely based on an acceleration of the beta1i/LMP2 and potentially of beta2i/MECL1 processing kinetics.     

The components of the proteasome system and their role in MHC class I antigen processing

By generating peptides from intracellular antigens which are then presented to T cells, the ubiquitin/26S proteasome system plays a central role in the cellular immune response. The proteolytic properties of the proteasome are adapted to the requirements of the immune system by proteasome components whose synthesis is under the control of interferon-γ. Among these are three subunits with catalytic sites that are incorporated into the enzyme complex during its de novo synthesis. Thus, the proteasome assembly pathway and the formation of immunoproteasomes play a critical regulatory role in the regulation of the proteasome's catalytic properties. In addition, interferon-γ also induces the synthesis of the proteasome activator PA28 which, as part of the so-called hybrid proteasome, exerts a more selective function in antigen presentation. Consequently, the combination of a number of regulatory events tunes the proteasome system to gain maximal efficiency in the generation of peptides with regard to their quality and quantity.     

Structure and Protein Composition of a Basal‐Body Scaffold (“Cage”) in the Hypotrich Ciliate Euplotes

Cilia on the ventral surface of the hypotrich ciliate Euplotes are clustered into polykinetids or compound ciliary organelles, such as cirri or oral membranelles, used in locomotion and prey capture. A single polykinetid may contain more than 150 individual cilia; these emerge from basal bodies held in a closely spaced array within a scaffold or framework structure that has been referred to as a basal‐body “cage”. Cage structures were isolated free of cilia and basal bodies; the predominant component of such cages was found on polyacrylamide gels to be a 45‐kDa polypeptide. Antisera were raised against this protein band and used for immunolocalizations at the light and electron microscope levels. Indirect immunofluorescence revealed the 45‐kDa polypeptide to be localized exclusively to the bases of the ventral polykinetids. Immunogold staining of thin sections of intact cells further localized this reactivity to filaments of a double‐layered dense lattice that appears to link adjoining basal bodies into ordered arrays within each polykinetid. Scanning electron microscopy of isolated cages reveals the lower or “basal” cage layer to be a fine lacey meshwork supporting the basal bodies at their proximal ends; adjoining basal bodies are held at their characteristic spacing by filaments of an upper or “medial” cage layer. The isolated cage thus resembles a miniature test‐tube rack, able to accommodate varying arrangements of basal‐body rows, depending on the particular type of polykinetid. Because of its clear and specific localization to the basal‐body cages in Euplotes, we have termed this novel 45‐kDa protein “cagein”.     

The N-terminal flanking region of the TRP2360-368 melanoma antigen determines proteasome activator PA28 requirement for epitope liberation

Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens. The interferon-gamma-inducible proteasome activator PA28 plays an important role in the generation of MHC ligands by proteasomes. Generation of the HLA-A(*)0201 restricted melanoma antigen TRP2(360-368) by the proteasome has been shown to be dependent on the function of PA28 in vitro and in vivo (Sun, Y., Sijts, A. J., Song, M., Janek, K., Nussbaum, A. K., Kral, S., Schirle, M., Stevanovic, S., Paschen, A., Schild, H., Kloetzel, P. M., and Schadendorf, D. (2002) Cancer Res. 62, 2875-2882). Here we analyzed the role of the epitope sequence environment in determining this PA28 dependence. Experiments using the melanoma TRP2(288-296) epitope and the murine cytomegalovirus-derived pp89 epitope precursor peptide for epitope replacement revealed that the TRP2(360-368) flanking sequences can transfer PA28 dependence onto otherwise PA28 independent epitopes. Moreover, the N-terminal flanking sequence is sufficient to establish PA28 dependence of an epitope by allowing PA28-induced coordinated dual cleavages. These results show that N-terminal flanking sequences strongly influence epitope generation efficiency and that PA28 function is particularly relevant for the generation of normally poorly excised peptide products.     

Immunoproteasome LMP2 60HH Variant Alters MBP Epitope Generation and Reduces the Risk to Develop Multiple Sclerosis in Italian Female Population

Background

Albeit several studies pointed out the pivotal role that CD4+T cells have in Multiple Sclerosis, the CD8+ T cells involvement in the pathology is still in its early phases of investigation. Proteasome degradation is the key step in the production of MHC class I-restricted epitopes and therefore its activity could be an important element in the activation and regulation of autoreactive CD8+ T cells in Multiple Sclerosis.

Methodology/Principal Findings

Immunoproteasomes and PA28-αβ regulator are present in MS affected brain area and accumulated in plaques. They are expressed in cell types supposed to be involved in MS development such as neurons, endothelial cells, oligodendrocytes, macrophages/macroglia and lymphocytes. Furthermore, in a genetic study on 1262 Italian MS cases and 845 controls we observed that HLA-A*02+ female subjects carrying the immunoproteasome LMP2 codon 60HH variant have a reduced risk to develop MS. Accordingly, immunoproteasomes carrying the LMP2 60H allele produce in vitro a lower amount of the HLA-A*0201 restricted immunodominant epitope MBP_111–119.

Conclusion/Significance

The immunoproteasome LMP2 60HH variant reduces the risk to develop MS amongst Italian HLA-A*02+ females. We propose that such an effect is mediated by the altered proteasome-dependent production of a specific MBP epitope presented on the MHC class I. Our observations thereby support the hypothesis of an involvement of immunoproteasome in the MS pathogenesis.     

Nuclear roles in the postconjugant development of the ciliated protozoan Euplotes aediculatus. 1. Evidence for sequential roles of the differentiating macronucleus in exconjugant development

Thin-section and freeze-fracture data retrieved from developing fetal adrenals of mouse, rabbit, and rat reveal that gap junctions which appear as cortical and medullary cells morphogenetically assort themselves. In the presumptive zona fasciculata and reticularis communicating junctions develop between neighboring cells just prior to the onset of steroidogenesis. In the rodents, gap junctions assemble in large, particle-free formation plaques 1 day before the proliferation of smooth endoplasmic reticulum and the vesiculation of mitochondria (morphological features which are indicative of steroidogenesis). Similar observations have been obtained in fetal rabbits, but here gap junctions apparently develop in the absence of formation plaques. In each species, gap junctions apparently develop 1 or 2 days prior to a surge in corticosteroid production, suggesting that the formation of communicating junctions during the early phases of adrenal cortical differentiation may provide an avenue to coordinate steroidogenesis in the developing fetal cortex.     

Structure and structure formation of the 20S proteasome

Eukaryotic 20S proteasomes are complex oligomeric proteins. The maturation process of the 14 different - and -subunits has to occur in a highly coordinate manner. In addition -subunits are synthesized as proproteins and correct processing has to be guaranteed during complex maturation. The structure formation can be subdivided in different phases. The knowledge of the individual phases is summarized in this publication. As a first step the newly synthesized monomers have to adopt the correct tertiary structure, a process that might be supported in the case of the -subunits by the intramolecular chaperone activity postulated for the prosequences. Subsequently the -subunits form ring-like structures thereby providing docking sites for the different -subunits. The result most likely is a double ring structure (13S precursor) representing half-proteasomes, which contain immature proproteins. Two 13S precursors associate to form the proteolytically inactive 16S assembly intermediate which still contains unprocessed -monomers. In addition the chaperone Hsc73 is present within these particles suggesting an essential role during the structure formation process. The processing of monomers with an N-terminal threonine occurs within the 16S particles and is achieved autocatalytically by two subsequent processing events finally leading to the mature, active 20S proteasome.