The murine cytomegalovirus pp89 immunodominant H-2Ld epitope is generated and translocated into the endoplasmic reticulum as an 11-mer precursor peptide

The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitopes is known to be generated as precursor peptides requiring trimming either before or after translocation into the endoplasmic reticulum (ER). In this study, we have followed the proteasomal processing and TAP-dependent ER translocation of the immunodominant epitope of the murine CMV immediate early protein pp89. For the first time, we experimentally linked peptide generation by the proteasome system and TAP-dependent ER translocation. Our experiments show that the proteasome generates both an N-terminally extended 11-mer precursor peptide as well as the correct H2-L(d) 9-mer epitope, a process that is accelerated in the presence of PA28. Our direct peptide translocation assays, however, demonstrate that only the 11-mer precursor peptide is transported into the ER by TAPs, whereas the epitope itself is not translocated. In consequence, our combined proteasome/TAP assays show that the 11-mer precursor is the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.     

Rearrangement of the 16S precursor subunits is essential for the formation of the active 20S proteasome

Proteasome-dependent proteolysis is essential for a number of key cellular processes and requires a sophisticated biogenesis pathway to function. Here, we have arrested the assembly process in its dynamic progression at the short-lived 16S state. Structural analysis of the 16S proteasome precursor intermediates by electron microscopy, and single particle analysis reveals major conformational changes in the structure of the beta-ring in comparison with one-half of the 20S proteasome. The individual beta-subunits in the 16S precursor complex rotate with respect to their positions in the x-ray crystallographic structure of the fully assembled 20S. This rearrangement results in a movement of the catalytic residue threonine-1 from the protected location in 16S precursor complexes to a more exposed position in the 20S structure. Thereby, our findings provide a molecular explanation for the structural rearrangements necessary for the dimerization of two 16S precursor complexes and the subsequent final maturation to active 20S proteasomes.     

Schistosomiasis in Brazil: Does social development suffice?

Up to 15 years ago, when asked to identify the major public health problems of Brazil, there would be no hesitation in naming infant diarrhoea, Chagas disease and Schistosoma mansoni. While most field workers have now eliminated schistosomiasis from the list, now certainly headed by malaria, in academic and bureaucratic circles the old priorities are still maintained.     

COMPARTMENTALIZATION OF THE DEVELOPING MACRONUCLEUS FOLLOWING CONJUGATION IN STYLONYCHIA AND EUPLOTES

The development of the macronucleus following conjugation in the hypotrichous ciliates Euplotes and Stylonychia has been examined with the electron microscope. Banded polytene chromosomes can be seen in thin sections of the macronuclear anlagen during the early periods of exconjugant development. As the chromosomes reach their maximum state of polyteny, sheets of fibrous material appear between the chromosomes and transect the chromosomes in the interband regions. Individual bands of the polytene chromosomes thus appear to be isolated in separate compartments. Subsequently, during the stage when the bulk of the polytenic DNA is degraded (1), these compartments swell, resulting in a nucleus packed with thousands of separate spherical chambers. Individual chromosomes are no longer discernible. The anlagen retain this compartmentalized condition for several hours, at the end of which time aggregates of dense material form within many of the compartments. The partitioning layers disperse shortly before replication bands appear within the elongating anlagen, initiating the second period of DNA synthesis characteristic of macronuclear development in these hypotrichs. The evidence presented here suggests that the "chromatin granules" seen in the mature vegetative macronucleus represent the material of single bands of the polytene chromosomes seen during the earlier stages of macronuclear development. The possibility is also discussed that the degradation of DNA in the polytene chromosomes may be genetically selective, which would result in a somatic macronucleus with a different genetic constitution than that of the micronucleus from which it was derived.     

The role of the proteasome system and the proteasome activator PA28 complex in the cellular immune response

The generation of antigenic peptides bound and presented to the immune system by MHC class I molecules predominantly depends on the function of the proteasome system. Stimulation of cells with interferon gamma induces the incorporation of three active site bearing beta-subunits into the 20S proteasome and the formation of the PA28 proteasome modulator complex. PA28 alters the cleavage properties of the proteasome and enhances MHC class I antigen presentation. Thus, by cytokine induced change of the proteasome system cells may alter the proteolytic properties of the 20S proteasome and may render an organism more flexible in its peptide generation capacity.     

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 ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway

To eliminate misfolded proteins that accumulate in the endoplasmic reticulum (ER) the cell mainly relies on ubiquitin-proteasome dependent ER-associated protein degradation (ERAD). Proteolysis of ERAD substrates by the proteasome requires their ubiquitylation and retro-translocation from the ER to the cytoplasm. Here we describe a high molecular mass protein complex associated with the ER membrane, which facilitates ERAD. It contains the ubiquitin domain protein (UDP) HERP, the ubiquitin protein ligase HRD1, as well as the retro-translocation factors p97, Derlin-1 and VIMP. Our data on the structural arrangement of these ERAD proteins suggest that p97 interacts directly with membrane-resident components of the complex including Derlin-1 and HRD1, while HERP binds directly to HRD1. We propose that ubiquitylation, as well as retro-translocation of proteins from the ER are performed by this modular protein complex, which permits the close coordination of these consecutive steps within ERAD.     

CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.