Homodimerization and hetero-oligomerization of the single-domain trefoil protein pNR-2/pS2 through cysteine 58.
ABSTRACT: The single-domain human trefoil proteins [pNR-2/pS2 and human intestinal trefoil factor (hITF)] have seven cysteine residues, of which six are involved in maintaining the structure of the trefoil domain. The seventh does not form part of the trefoil domain and is located three residues from the C-terminus. The ability of the pNR-2/pS2 single trefoil domain protein to dimerize was examined by using recombinant protein with either a cysteine or a serine residue at this position by equilibrium ultracentrifugation, laser-assisted desorption MS, gel filtration and PAGE. pNR-2/pS2 Cys58 formed dimers, whereas pNR-2/pS2 Ser58 did not. Experiments in which the dimer was treated with thiol agents demonstrated that the dimer was linked via a disulphide bond and that the intermolecular disulphide bond was more susceptible to reduction than the intramolecular disulphide bonds. To examine whether dimeric pNR-2/pS2 was secreted by oestrogen-responsive breast cancer cells, which are known to express pNR-2/pS2 mRNA, conditioned medium was separated on non-denaturing polyacrylamide gels, transferred to PVDF membrane and reacted with antiserum against pNR-2/pS2. Monomeric and dimeric pNR-2/pS2 were detected but the majority of the protein reactivity was associated with a larger protein. Treatment of this protein with thiol agents suggested that it is an oligomer containing pNR-2/pS2 linked to another protein by a disulphide bond. These studies suggest that the biological action of pNR-2/pS2 single-domain trefoil protein might involve the formation of homodimers or oligomers with other proteins.
Project description:The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the 'trefoil' family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2.
Project description:Many extracellular globular proteins have evolved to possess disulphide bonds in their native conformations, which aids in thermodynamic stabilisation. However, disulphide bond breakage by heating leads to irreversible protein denaturation through disulphide-thiol exchange reactions. In this study, we demonstrate that methanethiosulphonate (MTS) specifically suppresses the heat-induced disulphide-thiol exchange reaction, thus improving the heat-resistance of proteins. In the presence of MTS, small globular proteins that contain disulphides can spontaneously refold from heat-denatured states, maintaining wild-type disulphide pairing. Because the disulphide-thiol exchange reaction is triggered by the generation of catalytic amounts of perthiol or thiol, rapid and specific perthiol/thiol protection by MTS reagents prevents irreversible denaturation. Combining MTS reagents with another additive that suppresses chemical modifications, glycinamide, further enhanced protein stabilisation. In the presence of these additives, reliable remnant activities were observed even after autoclaving. However, immunoglobulin G and biotin-binding protein, which are both composed of tetrameric quaternary structures, failed to refold from heat-denatured states, presumably due to chaperon requirements. Elucidation of the chemical modifications involved in irreversible thermoinactivation is useful for the development of preservation buffers with optimum constitutions for specific proteins. In addition, the impact of disulphide bond breakage on the thermoinactivation of proteins can be evaluated using MTS reagents.
Project description:Photoreceptor-specific nuclear receptor (PNR/NR2E3) and Tailless homolog (TLX/NR2E1) are human orthologs of the NR2E group, a subgroup of phylogenetically related members of the nuclear receptor (NR) superfamily of transcription factors. We assessed the ability of these NRs to form heterodimers with other members of the human NRs representing all major subgroups. The TLX ligand-binding domain (LBD) did not appear to form homodimers or interact directly with any other NR tested. The PNR LBD was able to form homodimers, but also exhibited robust interactions with the LBDs of peroxisome proliferator-activated receptor-? (PPAR?)/NR1C3 and thyroid hormone receptor b (TRb) TR?/NR1A2. The binding of PNR to PPAR? was specific for this paralog, as no interaction was observed with the LBDs of PPAR?/NR1C1 or PPAR?/NR1C2. In support of these findings, PPAR? and PNR were found to be co-expressed in human retinal tissue extracts and could be co-immunoprecipitated as a native complex. Selected sequence variants in the PNR LBD associated with human retinopathies, or a mutation in the dimerization region of PPAR? LBD associated with familial partial lipodystrophy type 3, were found to disrupt PNR/PPAR? complex formation. Wild-type PNR, but not a PNR309G mutant, was able to repress PPAR?-mediated transcription in reporter assays. In summary, our results reveal novel heterodimer interactions in the NR superfamily, suggesting previously unknown functional interactions of PNR with PPAR? and TR? that have potential importance in retinal development and disease.
Project description:Emerging evidence has linked photoreceptor cell-specific nuclear receptor (PNR/NR2E3), an orphan nuclear hormone receptor, to human breast cancer. PNR was shown to be a transcriptional activator of estrogen receptor-? (ER?) in ER?-positive breast cancer cell lines and high-level expression of PNR correlates with favorable response of ER?-positive breast cancer patients to tamoxifen. Interestingly, gene expression microarray study shows that PNR regulates distinct genes from those regulated by ER?, suggesting that PNR could have ER?-independent functions. Herein, we investigated the function of PNR in ER?-negative breast cancer cells. Our results showed that PNR-induced cell migration and metastasis of ER?-negative breast cancer cells both in vitro and in vivo, and the effect was attributed to the upregulation of interleukin (IL)-13R?2, a high-affinity receptor for IL-13 that regulates tumor growth, invasion and metastasis of various human cancers. Mechanistically, PNR activated transcription of IL-13R?2 through direct recruitment to IL-13R?2 promoter. Upon stimulation with IL-13, IL-13R?2 increased the extracellular signal-regulated kinases 1 and 2 phosphorylation, which led to breast cancer migration and metastasis. The IL-13 triggered signal cascade was specific to IL-13R?2, as the closely related IL-13R?1 was not regulated by PNR. IL-13R?2 is a novel tumor antigen that is overexpressed in a variety of solid tumor types. This study presents the first evidence that PNR could promote ER?-negative breast cancer metastasis through activation of IL-13R?2-mediated signaling pathway.
Project description:The protein disulphide-bond isomerization activity of highly active homogeneous protein disulphide-isomerase (measured by re-activation of 'scrambled' ribonuclease) is enhanced by EDTA and by phosphate buffers. As shown for previous less-active preparations, the enzyme has a narrow pH optimum around pH 7.8 and requires the presence of either a dithiol or a thiol. The dithiol dithiothreitol is effective at concentrations 100-fold lower than the monothiols reduced glutathione and cysteamine. The enzyme follows Michaelis-Menten kinetics with respect to these substrates; Km values are 4,620 and 380 microM respectively. The enzyme shows apparent inhibition by high concentrations of thiol or dithiol compounds (greater than 10 X Km), but the effect is mainly on the extent of reaction, not the initial rate. This is interpreted as indicating the formation of significant amounts of reduced ribonuclease in these more reducing conditions. The purified enzyme will also catalyse net reduction of insulin disulphide bonds by reduced glutathione (i.e. it has thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase activity), but this requires considerably higher concentrations of enzyme and reduced glutathione than does the disulphide-isomerization activity. The Km for reduced glutathione in this reaction is an order of magnitude greater than that for the disulphide-isomerization activity, and the turnover number is considerably lower than that of other enzymes that can catalyse thiol-disulphide oxidoreduction. Conventional two-substrate steady-state analysis of the thiol:protein-disulphide oxidoreductase activity indicates that it follows a ternary-complex mechanism. The protein disulphide-isomerase and thiol:protein-disulphide oxidoreductase activities co-purify quantitatively through the final stages of purification, implying that a single protein species is responsible for both activities. It is concluded that previous preparations, from various sources, that have been referred to as protein disulphide-isomerase, disulphide-interchange enzyme, thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase are identical or homologous proteins. The assay, nomenclature and physiological role of this enzyme are discussed.
Project description:The BALB/c IgA (immunoglobulin A) myeloma protein M167 contained on average 5.7 free SH groups per IgA dimer. These groups were preponderantly on the heavy chains and comprised two distinct populations: 3.3 exposed SH groups per dimer in the Fc region, and 2.4 buried SH groups per dimer in the Fd region, detectable o only after denaturation. To locate the cysteine residues involved, labelled peptides were purified from thermolysin digests of radioalkylated IgA by high-performance liquid chromatography. From the amino acid compositions of the peptides, the exposed thiol groups were assigned to Cys-307 in the C alpha 2 domain, which thus existed in the reduced form to an extent exceeding 80%. This residue may allow attachment of secretory component to dimer IgA in the mouse to proceed via thiol-disulphide exchange. The buried thiol groups were assigned to Cys-150 and Cys-208, in the C alpha 1 domain, each being in the reduced form to the extent of approx. 30%. This pair of residues would normally give rise to the characteristic intradomain disulphide bridge. It appears that disulphide formation is not a crucial event during folding of the C alpha 1 domain in IgA biosynthesis. The sequence in the region 140-151 was re-investigated, and residue 142 was shown to be serine, not cysteine, helping explain the lack of heavy-chain-light chain bonding in BALB/c mouse IgA. A disulphide-bond model for mouse IgA is proposed on the basis of these assignments and other features of the mouse alpha-chain sequence.
Project description:A decapeptide containing two cysteine residues at positions 3 and 8 has been designed for use in monitoring the disulphide bond-forming activity of thiol:disulphide oxidoreductases. The peptide contains a tryptophan residue adjacent to one of the cysteine residues and an arginine residue adjacent to the other. Oxidation of this dithiol peptide to the disulphide state is accompanied by a significant change in tryptophan fluorescence emission intensity. This fluorescence quenching was used as the basis for monitoring the disulphide bond-forming activity of the enzymes protein disulphide-isomerase (PDI) and DsbA (a periplasmic protein thiol:disulphide oxidoreductase) in the pH range 4.0-7.5, where the rates of spontaneous or chemical oxidation are low. Reaction rates were found to be directly proportional to enzyme concentration, and more detailed analysis indicated that the rate-determining step in the overall process was the reoxidation of the reduced form of the enzyme by GSSG. The pH-dependence of the enzyme-catalysed reaction reflected primarily the pKa of the reactive cysteine residue at the active site of each enzyme. The data indicate a pKapp of 5.6 for bovine PDI and of 5.1 for Vibrio cholerae DsbA.
Project description:Photoreceptor-specific nuclear receptor (PNR, NR2E3) is a key transcriptional regulator of human photoreceptor differentiation and maintenance. Mutations in the NR2E3-encoding gene cause various retinal degenerations, including Enhanced S-cone syndrome, retinitis pigmentosa, and Goldman-Favre disease. Although physiological ligands have not been identified, it is believed that binding of small molecule agonists, receptor desumoylation, and receptor heterodimerization may switch NR2E3 from a transcriptional repressor to an activator. While these features make NR2E3 a potential therapeutic target for the treatment of retinal diseases, there has been a clear lack of structural information for the receptor. Here, we report the crystal structure of the apo NR2E3 ligand binding domain (LBD) at 2.8 Å resolution. Apo NR2E3 functions as transcriptional repressor in cells and the structure of its LBD is in a dimeric auto-repressed conformation. In this conformation, the putative ligand binding pocket is filled with bulky hydrophobic residues and the activation-function-2 (AF2) helix occupies the canonical cofactor binding site. Mutations designed to disrupt either the AF2/cofactor-binding site interface or the dimer interface compromised the transcriptional repressor activity of this receptor. Together, these results reveal several conserved structural features shared by related orphan nuclear receptors, suggest that most disease-causing mutations affect the receptor's structural integrity, and allowed us to model a putative active conformation that can accommodate small ligands in its pocket.
Project description:The membrane fusion activity of murine leukaemia virus Env is carried by the transmembrane (TM) and controlled by the peripheral (SU) subunit. We show here that all Env subunits of the virus form disulphide-linked SU-TM complexes that can be disrupted by treatment with NP-40, heat or urea, or by Ca(2+) depletion. Thiol mapping indicated that these conditions induced isomerization of the disulphide-bond by activating a thiol group in a Cys-X-X-Cys (CXXC) motif in SU. This resulted in dissociation of SU from the virus. The active thiol was hidden in uninduced virus but became accessible for alkylation by either Ca(2+) depletion or receptor binding. The alkylation inhibited isomerization, virus fusion and infection. DTT treatment of alkylated Env resulted in cleavage of the SU-TM disulphide-bond and rescue of virus fusion. Further studies showed that virus fusion was specifically inhibited by high and enhanced by low concentrations of Ca(2+). These results suggest that Env is stabilized by Ca(2+) and that receptor binding triggers a cascade of reactions involving Ca(2+) removal, CXXC-thiol exposure, SU-TM disulphide-bond isomerization and SU dissociation, which lead to fusion activation.
Project description:Photoreceptor cell-specific receptor (PNR/NR2E3) is an orphan nuclear receptor that plays a critical role in retinal development and photoreceptor maintenance. The disease-causing mutations in PNR have a pleiotropic effect resulting in varying retinal diseases. Recently, PNR has been implicated in control of cellular functions in cancer cells. PNR was reported to be a novel regulator of ER? expression in breast cancer cells, and high PNR expression correlates with favorable response to tamoxifen treatment. Moreover, PNR was shown to increase p53 stability in HeLa cells, implying that PNR may be a therapeutic target in this and other cancers that retain a wild type p53 gene. To facilitate further understanding of PNR functions in cancer, we characterized compound 11a, a synthetic, putative PNR agonist in several cell-based assays. Interestingly, we showed that 11a failed to activate PNR and its cytotoxicity was independent of PNR expression, excluding PNR as a mediator for 11a cytotoxicity. Systematic analyses of the cytotoxic effects of 11a in NCI-60 cell lines revealed a strong positive correlation of cytotoxicity with p53 status, i.e., p53 wild type cell lines were significantly more sensitive to 11a than p53 mutated or null cell lines. Furthermore, using HCT116 p53+/+ and p53-/- isogenic cell lines we revealed that the mechanism of 11a-induced cytotoxicity occurred through G1/S phase cell cycle arrest rather than apoptosis. In conclusion, we observed a correlation of 11a sensitivity with p53 status but not with PNR expression, suggesting that tumors expressing wild type p53 might be responsive to this compound.