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

Abstract 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 …

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 …

Solution Structure of the R3H Domain from Human Sμbp-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 o…

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. …

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 relaxat…

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

AbstractThe 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 t…

nmr structure of the pyrin domain of human asc