Proteasomal degradation of core protein variants from chronic hepatitis B patients.

The accumulation of complex hepatitis B virus (HBV) variants with internal in-frame deletions in the C gene in immunosuppressed renal transplant recipients is associated with a severe course of the infection leading to end-stage liver disease (ESLD). A set of six HBV C genes with internal in-frame deletions corresponding to the pattern of HBV population in immunosuppressed patients has been expressed in two different eukaryotic cell lines. Synthesis and proteasomal degradation of HBV core (HBc) protein variants were compared with those of the wild-type HBc. In all cases, the steady-state level of internally deleted HBc proteins, predominantly with longer deletions, were considerably lower and turnover was significantly higher in comparison with those of the wild-type HBc, since all deletion variants were degraded rapidly via the proteasome pathway. Involvement and consequences of the proteasomal degradation machinery in the HBc protein turnover during HBV infection with complex HBV variants in the immunosuppressed patients are discussed.

Site-specific labelling with a metal chelator for protein-structure refinement.

A single free Cys sidechain in the N-terminal domain of the E. coli arginine repressor was covalently derivatized with S-cysteaminyl-EDTA for site-specific attachment of paramagnetic metal ions. The effects of chelated metal ions were monitored with (15)N-HSQC spectra. Complexation of Co(2+), which has a fast relaxing electron spin, resulted in significant pseudocontact shifts, but also in peak doubling which was attributed to the possibility of forming two different stereoisomers of the EDTA-Co(2+) complex. In contrast, complexation of Cu(2+) or Mn(2+), which have slowly relaxing electron spins, did not produce chemical shift changes and yielded self-consistent sets of paramagnetic relaxation enhancements of the amide protons. T (1) relaxation enhancements with Cu(2+) combined with T (2) relaxation enhancements with Mn(2+) are shown to provide accurate distance restraints ranging from 9 to 25 A. These long-range distance restraints can be used for structural studies inaccessible to NOEs. As an example, the structure of a solvent-exposed loop in the N-terminal domain of the E. coli arginine repressor was refined by paramagnetic restraints. Electronic correlation times of Cu(2+) and Mn(2+) were determined from a comparison of T (1) and T (2) relaxation enhancements.

The death-domain fold of the ASC PYRIN domain, presenting a basis for PYRIN/PYRIN recognition.

The PYRIN domain is a conserved sequence motif identified in more than 20 human proteins with putative functions in apoptotic and inflammatory signalling pathways. The three-dimensional structure of the PYRIN domain from human ASC was determined by NMR spectroscopy. The structure determination reveals close structural similarity to death domains, death effector domains, and caspase activation and recruitment domains, although the structural alignment with these other members of the death-domain superfamily differs from previously predicted amino acid sequence alignments. Two highly positively and negatively charged surfaces in the PYRIN domain of ASC result in a strong electrostatic dipole moment that is predicted to be present also in related PYRIN domains. These results suggest that electrostatic interactions play an important role for the binding between PYRIN domains. Consequently, the previously reported binding between the PYRIN domains of ASC and ASC2/POP1 or between the zebrafish PYRIN domains of zAsc and Caspy is proposed to involve interactions between helices 2 and 3 of one PYRIN domain with helices 1 and 4 of the other PYRIN domain, in analogy to previously reported homophilic interactions between caspase activation and recruitment domains.

Solution structure of the R3H domain from human Smubp-2.

The R3H domain is a conserved sequence motif, identified in over 100 proteins, that is thought to be involved in polynucleotide-binding, including DNA, RNA and single-stranded DNA. In this work the 3D structure of the R3H domain from human Smubp-2 was determined by NMR spectroscopy. It is the first 3D structure determination of an R3H domain. The fold presents a small motif, consisting of a three-stranded antiparallel beta-sheet and two alpha-helices, which is related to the structures of the YhhP protein and the C-terminal domain of the translational initiation factor IF3. The similarities are non-trivial, as the amino acid identities are below 10%. Three conserved basic residues cluster on the same face of the R3H domain and could play a role in nucleic acid recognition. An extended hydrophobic area at a different site of the molecular surface could act as a protein-binding site. A strong correlation between conservation of hydrophobic amino acids and side-chain solvent protection indicates that the structure of the Smubp-2 R3H domain is representative of R3H domains in general.

Inhibition of ubiquitin-dependent proteolysis by a synthetic glycine-alanine repeat peptide that mimics an inhibitory viral sequence.

The glycine-alanine repeat (GAr) of the Epstein-Barr virus nuclear antigen-1 is a cis-acting transferable element that inhibits ubiquitin/proteasome-dependent proteolysis in vitro and in vivo. We have here examined the effect of a synthetic 20-mer GAr oligopeptide on the degradation of iodinated or biotin labeled lysozyme in a rabbit reticulocyte lysates in vitro assay. Micromolar concentrations of the GA-20 peptide inhibited the hydrolysis of lysozyme without significant effect on ubiquitination. Addition of the peptide did not inhibit the hydrolysis of fluorogenic substrate by purified proteasomes and did not affect the ubiquitination of lysozyme. An excess of the peptide failed to compete for binding of a synthetic tetra-ubiquitin complex to the S5a ubiquitin-binding subunit of the 19S regulator, confirming that the GAr does not block the access of ubiquitinated substrates to the proteasome. Our data suggest that the GAr may act by destabilizing the interaction of ubiquitinated substrates with the proteasome and promote the premature release of the substrate.

Genomic organization and promoter characterization of the gene encoding a putative endoplasmic reticulum chaperone, ERp29.

ERp29 is a soluble protein localized in the endoplasmic reticulum (ER) of eukaryotic cells, which is conserved in all mammalian species. The N-terminal domain of ERp29 displays sequence and structural similarity to the protein disulfide isomerase despite the lack of the characteristic double cysteine motif. Although the exact function of ERp29 is not yet known, it was hypothesized that it may facilitate folding and/or export of secretory proteins in/from the ER. ERp29 is induced by ER stress, i.e. accumulation of unfolded proteins in the ER. To gain an insight into the mechanisms regulating ERp29 expression we have cloned and characterized the rat ERp29 gene and studied in details its distribution in human tissues. Comparison with the murine and human genes and phylogenetic analysis demonstrated common origin and close ortholog relationships of these genes. Additionally, we have cloned approximately 3 kb of the 5'-flanking region of the ERp29 gene and functionally characterized its promoter. Such characteristics of the promoter as GC-rich sequence, absence of TATA-box, multiple transcription start sites taken together with the ubiquitous gene expression, reaching maximum levels in the specialized secretory tissues, indicate that ERp29 belongs to the group of the constitutively expressed housekeeping genes. A 337 bp fragment of the 5' flank was identified as a core promoter sufficient for the transcriptional activation of the gene. Gel mobility shift assay indicated interaction of the predicted GC and E box elements within the core promoter with Sp1/Sp3 and USF1/USF2 transcription factors, respectively, suggesting their key role in the basal expression of the gene.

Identification of ERp29, an endoplasmic reticulum lumenal protein, as a new member of the thyroglobulin folding complex.

Folding and post-translational modification of the thyroid hormone precursor, thyroglobulin (Tg), in the endoplasmic reticulum (ER) of the thyroid epithelial cells is facilitated by several molecular chaperones and folding enzymes, such as BiP, GRP94, calnexin, protein disulfide isomerase, ERp72, and others. They have been shown to associate simultaneously and/or sequentially with Tg in the course of its maturation, thus forming large heterocomplexes in the ER of thyrocytes. Here we present evidence that such complexes include a novel member, an ER-resident lumenal protein, ERp29, which is present in all mammalian tissues with exceptionally high levels of expression in the secretory cells. ERp29 was induced upon treatment of FRTL-5 rat thyrocytes with the thyroid-stimulating hormone, which is essential for the maintenance of thyroid cells and Tg biosynthesis. Chemical cross-linking followed by the cell lysis and immunoprecipitation of ERp29 or Tg revealed association of these proteins and additionally, immunocomplexes that also included major ER chaperones, BiP and GRP94. Sucrose density gradient analysis indicated co-localization of ERp29 with Tg and BiP in the fractions containing large macromolecular complexes. This was supported by immunofluorescent microscopy showing co-localization of ERp29 with Tg in the putative transport vesicular structures. Affinity chromatography using Tg as an affinity ligand demonstrated that ERp29 might be selectively isolated from the FRTL-5 cell lysate or purified lumenal fraction of rat liver microsomes along with the other ER chaperones. Preferential association with the urea-denatured Tg-Sepharose was indicative of either direct or circuitous ERp29/Tg interactions in a chaperone-like manner. Despite the presence of the C-terminal ER-retrieval signal, significant amounts of ERp29 were also recovered from the culture medium of stimulated thyrocytes, indicating ERp29 secretion. Based on these data, we suggest that the function of ERp29 in thyroid cells is connected with folding and/or secretion of Tg.

The Haemophilus ducreyi cytolethal distending toxin activates sensors of DNA damage and repair complexes in proliferating and non-proliferating cells.

Cytolethal distending toxins (CDTs) block proliferation of mammalian cells by activating DNA damage-induced checkpoint responses. We demonstrate that the Haemophilus ducreyi CDT (HdCDT) induces phosphorylation of the histone H2AX as early as 1 h after intoxication and re-localization of the DNA repair complex Mre11 in HeLa cells with kinetics similar to those observed upon ionizing radiation. Early phosphorylation of H2AX was dependent on a functional Ataxia Telangiectasia mutated (ATM) kinase. Microinjection of a His-tagged HdCdtB subunit, homologous to the mammalian DNase I, was sufficient to induce re-localization of the Mre11 complex 1 h post treatment. However, the enzymatic potency was much lower than that exerted by bovine DNase I, which caused marked chromatin changes at 106 times lower concentrations than HdCdtB. H2AX phosphorylation and Mre11 re-localization were induced also in HdCDT-treated, non-proliferating dendritic cells (DCs) in a differentiation dependent manner, and resulted in cell death. The data highlight several novel aspects of CDTs biology. We demonstrate that the toxin activates DNA damage-associated molecules in an ATM-dependent manner, both in proliferating and non-proliferating cells, acting as other DNA damaging agents. Induction of apoptotic death of immature DCs by HdCDT may represent a previously unknown mechanism of immune evasion by CDT-producing microbes.

Functional p53 chimeras containing the Epstein-Barr virus Gly-Ala repeat are protected from Mdm2- and HPV-E6-induced proteolysis.

Functional inactivation of the tumor suppressor protein p53 by accelerated ubiquitin/proteasome-dependent proteolysis is a common event in tumor progression. Proteasomal degradation is inhibited by the Gly-Ala repeat (GAr) of the Epstein-Barr virus nuclear antigen-1, which acts as a transferable element on a variety of proteasomal substrates. We demonstrate that p53 chimeras containing GAr domains of different lengths and positions within the protein are protected from proteolysis induced by the ubiquitin ligases murine double minute 2 and E6-associated protein but are still ubiquitinated and retain the capacity to interact with the S5a ubiquitin-binding subunit of the proteasome. The GAr chimeras transactivate p53 target genes, induce cell cycle arrest and apoptosis, and exhibit improved growth inhibitory activity in tumor cells with impaired endogenous p53 activity.

In vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein.

A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH(2)-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures approximately 14 degrees C higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH(2) and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (Delta Delta G approximately 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.