Structure of an XRCC1 BRCT domain: a new protein-protein interaction module.

The BRCT domain (BRCA1 C-terminus), first identified in the breast cancer suppressor protein BRCA1, is an evolutionarily conserved protein-protein interaction region of approximately 95 amino acids found in a large number of proteins involved in DNA repair, recombination and cell cycle control. Here we describe the first three-dimensional structure and fold of a BRCT domain determined by X-ray crystallography at 3.2 A resolution. The structure has been obtained from the C-terminal region of the human DNA repair protein XRCC1, and comprises a four-stranded parallel beta-sheet surrounded by three alpha-helices, which form an autonomously folded domain. The compact XRCC1 structure explains the observed sequence homology between different BRCT motifs and provides a framework for modelling other BRCT domains. Furthermore, the established structure of an XRCC1 BRCT homodimer suggests potential protein-protein interaction sites for the complementary BRCT domain in DNA ligase III, since these two domains form a stable heterodimeric complex. Based on the XRCC1 BRCT structure, we have constructed a model for the C-terminal BRCT domain of BRCA1, which frequently is mutated in familial breast and ovarian cancer. The model allows insights into the effects of such mutations on the fold of the BRCT domain.

Crystallization of an intact GST-estrogen receptor hormone binding domain fusion protein.

Crystals of an intact GST-estrogen receptor hormone binding domain fusion protein have been grown from solutions of MPD. The crystals grew as clusters of thin plates and needles of maximum dimensions 100 x 20 x 1 micrometer but were unsuitable for X-ray diffraction analysis. However, examination by electron microscopy shows an ordered lattice in which the protein molecules are clearly visible. Image analysis of electron micrographs of the protein crystals revealed electron stain-excluding density which showed a two-domain trimeric structure in projection, with each molecule of dimensions 12.0 x 5.0 nm diameter. The use of GST-fusion proteins in crystallisation are discussed.

Open complex formation around a lesion during nucleotide excision repair provides a structure for cleavage by human XPG protein.

Human XPG nuclease makes the 3' incision during nucleotide excision repair of DNA. The enzyme cleaves model DNA bubble structures specifically near the junction of unpaired DNA with a duplex region. It is not yet known, however, whether an unpaired structure is an intermediate during actual DNA repair. We find here that XPG requires opening of >5 bp for efficient cleavage. To seek direct evidence for formation of an open structure around a lesion in DNA during a nucleotide excision repair reaction in vitro, KMnO4 footprinting experiments were performed on a damaged DNA molecule bearing a uniquely placed cisplatin adduct. An unwound open complex spanning approximately 25 nucleotides was observed that extended to the positions of 5' and 3' incision sites and was dependent on XPA protein and on ATP. Opening during repair occurred prior to strand incision by XPG.

Biochemical characterization and novel isolation of pure estrogen receptor hormone-binding domain.

Biologically active, mouse estrogen receptor hormone-binding domain (residues 313-599) overexpressed in Escherichia coli was purified to apparent homogeneity as a single component with a molecular mass of 32.831 kDa determined by electrospray ionization mass spectrometry, and was identical to the mass predicted from the amino acid sequence. The intact domain was isolated using a novel, rapid purification scheme without recourse to any chromatographic process. Pure ERhbd maintained both high affinity estradiol binding (at optimum pH 8.0) and specificity for estrogens and anti-estrogens. The steroid-binding domain sedimented as a 4S component in the presence or absence of bound [3H]estradiol and at 2S in the presence of urea. The molecular mass of the 4S steroid unoccupied ERhbd (from dynamic light scattering) was approximately 72 kDa, suggesting that the pure, unlabelled ERhbd formed homodimers. Steroid-labelled ERhbd electrofocussed as a single, acidic component at a pI of 5.6. Binding of ERhbd to [3H]estradiol was unaffected by Ca2+ and Mg2+ ions up to 1 mM but was significantly inhibited by Zn2+ ions at concentrations above 10 microM, an effect reversed by EDTA.

20S human proteasomes bind with a specific orientation to lipid monolayers in vitro.

20S Proteasomes are non-lysosomal, high molecular weight proteinases implicated in the degradation of misfolded proteins and several short-lived regulatory proteins. They have a well established role, as the core of the 26S proteasome complex, in the ubiquitin-dependent proteolytic pathway and in antigen processing. While correctly folded proteins are not degraded by the 20S proteasome, unfolding, for example by oxidation, may render them degradable. The 20S proteasome is a 700-kDa cylindrical particle, composed of 14 subunits of molecular masses 20-35 kDa. There is evidence that 20S proteasomes are in close proximity to or associate with the endoplasmic reticulum and nuclear and plasma membranes in vivo. To better understand the lipid association of 20S proteasomes in vitro, we used a lipid monolayer system as a simple model system for biological membranes. The structure and orientation of the monolayer lipid bound 20S proteasomes has been determined by electron microscopy. 20S proteasomes associated in an "end-on' configuration specifically on PI lipid monolayers forming large arrays, with their channels opposite the lipid headgroups. On ER and Golgi lipid films 20S proteasomes were oriented in the same way as on the PI lipid film but were monodisperse. Protein molecules were randomly oriented in the presence of PA, PG, PS, PC and mitochondrial lipid monolayers. We show that 20S proteasomes bind to phospholipids in vitro in a preferred orientation which places the proteasome channel perpendicular to the membrane.

Divalent metal ions induce conformational change in pure, human wild-type p53 tumor suppressor protein.

The ability of wild-type and not mutant p53 to exert antiproliferative effects on normal cells may be related to a difference in the conformational state of the protein. We have used pure, human wild-type p53 and a panel of monoclonal antibodies whose epitopes map throughout the protein to assess whether divalent metal ions affect the conformation of p53. Our results show that the presence of Zn2+ ions at physiological concentrations, directly reduced or blocked accessibility of epitopes on pure wild-type p53, an effect which was reversed by chelating agents. Loss of epitope reactivity was maximal between the protein mid-region and C-terminus. Analytical sucrose density gradient ultracentrifugation studies also confirmed that Zn(2+)-induced conformational changes partially affected the pattern of p53 oligomerisation. The observed binding of pure p53 to a sequence-specific DNA motif was unaffected by the presence of added Zn2+ ions or metal chelating agents.

Biochemical characterisation of purified human wild-type p53 overexpressed in insect cells.

Conditions for the overexpression of human wild-type p53 using a baculovirus construct were optimised in insect cells which produced up to 20 mg p53/1 culture. Milligram amounts of p53 were purified to apparent homogeneity using chromatography on double-stranded DNA-cellulose (approximately 58% yield) followed by immunoaffinity chromatography with an epitope elution step (up to 48% yields) at 4 degrees C. The M(r) of extracted p53 both from insect cell lysates and after purification was 54,000 by SDS/PAGE. Isoelectric focusing showed recombinant p53 to be an acidic protein, focusing at pI 6.0 under non-denaturing conditions. Expressed p53 at all stages of purification reacted by immunoblotting with specific p53 monoclonal antibodies, indicating the presence of intact epitopes at the C-terminus, N-terminus and central region of the protein. From ultracentrifugation studies, pure p53 exhibited significant oligomerisation, and sedimented broadly within the 7-12-S region of sucrose gradients. Pure p53 slowly precipitated out of solution at concentrations between 1-6 mg/ml even in the presence of 1% detergent. Using metal affinity chromatography, we have established that pure p53 binds the immobilised divalent ions Zn2+, Ni2+ and Co2+ with high affinity.

Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth.

Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene. Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit. HGF binding to endothelial cells identifies two sites with different affinities. The higher affinity binding site (Kd = 0.35 nM) corresponds to the p190MET receptor. Sub-nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility. HGF induces repairs of a wound in endothelial cell monolayer. HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype. In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations. HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions. These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate.

Presence of an oestradiol receptor-related protein in the skin: changes during the normal menstrual cycle.

OBJECTIVE: To investigate the presence of an oestradiol receptor-related protein (P29) in skin and skin organelles, and to assess changes in its content during the normal menstrual cycle. DESIGN: An observational study. SETTING: King's College School of Medicine and Dentistry, London. SUBJECTS: Twenty-one premenopausal women with regular menstrual cycles undergoing gynaecological surgery. They were allocated to proliferative or secretory phases of the menstrual cycle on the basis of menstrual dating and histological examination of an endometrial sample. INTERVENTIONS: Small full thickness sections of skin (about 5 mm in depth) taken from the anterior abdominal wall at hysterectomy or laparoscopic sterilization. MAIN OUTCOME MEASURES: The concentration of the oestradiol receptor-related protein in skin and its organelles was assessed semi-quantitatively, using a monoclonal antibody technique. The intensity of staining was compared between the proliferative and secretory phases of the cycle. RESULTS: The receptor-related protein was consistently observed in epidermis, sebaceous glands, hair follicles and sweat ducts; there was no significant difference in its concentration between the proliferative and secretory phases of the menstrual cycle. The protein was not present in dermis and sweat ducts. CONCLUSIONS: Epidermis and some skin organelles contain an oestradiol receptor-related protein and must be considered as oestrogen target tissues. However, the content of this protein does not appear to change significantly during the normal menstrual cycle.

Purification and characterization of biologically active scatter factor from ras-transformed NIH 3T3 conditioned medium.

Scatter factor (SF), a glycoprotein produced by cultured fibroblasts, acts in vitro on epithelial cells causing separation and increased local motility. In this study, the polypeptide was purified to apparent homogeneity in high yields with conserved biological activity from medium conditioned by ras-transformed NIH 3T3 cells, by a three-step procedure involving ammonium sulphate fractionation, cation-exchange and hydroxyapatite chromatography. After purification, SF specific activity increased from approximately 0.3 units/microgram in unprocessed conditioned medium to approximately 5 units/ng, and cumulative recovery of biological activity was approximately 38%. Treatment of pure SF with N-glycanase resulted in a decreased Mr, but no concomitant effect was observed on biological activity. Proteolytic activity was absent from samples of both partially purified and pure SF. Our biochemical studies showed that SF, which is highly aggregated in low-ionic-strength media, is not aggregated in 0.4 M-salt. Under non-reducing conditions, pure SF migrated as a single stained band at Mr 67,000 on SDS/PAGE, and biological activity was eluted from unstained gels with an identical Mr. SF was electrofocused sharply at pI 8.5 with no degradation of activity. From ultracentrifugation studies (under non-aggregating conditions), the sedimentation coefficient of active SF was 3.7 S and f.p.l.c. molecular sieve chromatography indicated a Stokes' radius of 2.95 nm. The calculated Mr from these data was 61,400. The appearance of three stained polypeptides of Mr 82,000, 57,000 and 32,000 derived from the Mr-67,000 constituent after reduction with mercaptoethanol suggests that SF may be a heterodimer of Mr-57,000 and -32,000 subunits. Data from protein sequence analysis of the hydroxyapatite-purified protein confirms that SF has sequence identity with both rat hepatocyte growth factor and human fibroblast tumour cytotoxic factor.

Purification and characterization of scatter factor.

Scatter factor is a fibroblast-derived protein which disrupts and scatters epithelial colonies and enhances the local movement of individual epithelial and endothelial cells. The factor purified from mouse fibroblasts by cation-exchange and reverse phase chromatography is a dimer of 57 kD and 30 kD protein subunits (A and B subunits), is active at picomolar concentrations and requires intact intra- and/or inter-chain disulphide bonds for activity. In serum-free conditioned medium the factor is highly aggregated but in the presence of high-salt buffers or protein denaturants elutes from gel filtration columns with an apparent Mr of approximately 50 kD. From a combination of molecular sieving and ultracentrifugation studies, a calculated Mr of 61.4 kD is obtained for native mouse scatter factor, a value which agrees well with the Mr estimates obtained by SDS-PAGE (62-67 kD). Mouse fibroblast scatter factor is a heparin-binding, basic protein (pI 8.5-9.5) which contains N-linked carbohydrates which are not, however, essential for activity. The factor has no metallo- or serine protease activity and there is no evidence so far that its junctional-breaking activity involves proteolytic cleavage of surface molecules on target cells. Scatter factor is either identical or closely related to hepatocyte growth factor/hepatopoietin A (a potent mitogen for rat hepatocytes recently purified from human and rabbit serum and rat platelets). The factor is thus an effector of mesenchymal-epithelial interactions which affects the movement or the growth of different epithelia.

Bombesin receptor from Swiss 3T3 cells. Affinity chromatography and reconstitution into phospholipid vesicles.

Bombesin and its mammalian counterpart gastrin releasing peptide (GRP) are potent mitogens for Swiss 3T3 cells in which distinct high affinity receptors have been identified. We developed here a probe for specific ligand affinity chromatography by coupling biotin to [lys3]bombesin. The resulting biotinylated [lys3]bombesin (BLB) retained biological activity as judged by inhibition of [125I]GRP binding to intact cells and membrane preparations and stimulation of rapid Ca2+ mobilization and DNA synthesis in intact cells. Using this ligand and magnetised beads coated with streptavidin, we extracted differentially a single protein from detergent-solubilized Swiss 3T3 membranes in a BLB-dependent manner. Visualization was achieved either after autoradiograph of metabolically labelled proteins with [35S]methionine or by silver staining of larger preparations. In other experiments, elution of BLB-receptor complexes bound to streptavidin beads was carried out at neutral pH and the eluted fraction was reconstituted into phospholipid vesicles. This procedure revealed [125I]GRP binding activity that exhibited saturability, specificity and a 1946-fold increase in specific activity.

Immunoaffinity purification and characterisation of p29--an estrogen receptor related protein.

p29, a 29 kDa protein recognised by D5, a monoclonal antibody prepared against partially purified cytosolic estrogen receptor (ER), has been purified to homogeneity from ZR-75-1, a human breast cancer cell line. Ammonium sulphate fractionation followed by immunoaffinity chromatography on a three column system using Protein A-Sepharose coupled D5, produced purified p29. Silver stained SDS one-dimensional polyacrylamide gel electrophoresis (PAGE) and two-dimensional PAGE showed p29 to have been purified to homogeneity. Amino acid analysis showed no unusual characteristics. Partial N-terminal sequencing studies showed that purified p29 shared a 100% homology with the sequence of a pp89, murine cytomegaloviral protein.

Solubilization of the bombesin receptor from Swiss 3T3 cell membranes. Functional association to a guanine nucleotide regulatory protein.

Bombesin and structurally related peptides including gastrin releasing peptide (GRP) are potent mitogens for Swiss 3T3 cells. Here we attempted to solubilize bombesin receptors under conditions in which the ligand (125I-labelled GRP) was prebound to the receptor prior to detergent extraction. We found that 125I-GRP-receptor complexes were solubilized from Swiss 3T3 cell membranes by using the detergents taurodeoxycholate or deoxycholate. These detergents promoted ligand-receptor solubilization in a dose-dependent manner. In contrast, a variety of other detergents including Triton X-100, octylglycoside, CHAPS, digitonin, cholic acid and n-dodecyl-beta-D-maltoside, were much less effective. Addition of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) to ligand-receptor complexes isolated by gel filtration enhanced the rate of ligand dissociation in a concentration-dependent and nucleotide-specific manner. Our results demonstrate for the first time the successful solubilization of 125I-GRP-receptor complexes from Swiss 3T3 cell membranes and provide evidence for the physical association between the ligand-receptor complex and a guanine nucleotide binding protein(s).

Mitogenic signalling through the bombesin receptor: role of a guanine nucleotide regulatory protein.

Bombesin and structurally related peptides including gastrin releasing peptide (GRP) are potent mitogens for Swiss 3T3 cells. The early cellular and molecular responses elicited by bombesin and structurally related peptides have been elucidated in detail. Further understanding of the molecular basis of the potent mitogenic response initiated by bombesin is required in order to elucidate the mechanism by which the occupied receptor communicates with effector molecules in the cell. Transmembrane signalling mechanisms involving either a tyrosine kinase or a guanine nucleotide-binding regulatory protein (G protein) have been proposed. Here we summarize our experimental evidence indicating that a G protein(s) is involved in the coupling of the bombesin receptor to the generation of intracellular signals related to mitogenesis. Evidence for the role of G proteins in bombesin signal transduction pathways has been obtained by assessing the effects of guanine nucleotide analogues on both receptor-mediated responses in permeabilized cells and ligand binding in membrane preparations. We found that [125I]GRP-receptor complexes were solubilized from Swiss 3T3 cell membranes by using the detergents taurodeoxycholate or deoxycholate. Addition of guanosine 5-[gamma-thio]triphosphate (GTP gamma S) to ligand-receptor complexes isolated by gel filtration enhanced the rate of ligand dissociation in a concentration-dependent and nucleotide-specific manner. These results demonstrate the successful solubilization of [125I]GRP-receptor complexes from Swiss 3T3 cell membranes and provide evidence for the physical association between the ligand-receptor complex and a guanine nucleotide binding protein(s).

Characterization of p29, an estrogen-receptor associated tumor marker.

Monoclonal antibody D5, raised against cytosolic human estrogen receptor (ER) reacts with p29, a receptor-associated cytoplasmic serine phosphoprotein which does not bind steroid, While p29 selectively binds GTP and to a lesser extent ATP, in vitro GTP binding does not result in p29 phosphorylation. Under ER activating conditions, p29 associates with cytosolic ER; GTP, ATP and sodium molybdate block formation of immunoprecipitable p29-ER complexes. Nucleotide binding data suggest a role for p29 in the estrogen response machinery, possibly at the level of phosphate or nucleotide metabolism.

Immunohistochemical study of cytoplasmic oestradiol receptor in normal, dysplastic and malignant cervical tissue.

Using a monoclonal antibody raised against an oestrogen receptor-related protein, p29, and an indirect immunoperoxidase technique to stain human tissue, the presence of the antigen was investigated in normal, dysplastic and malignant tissue of the uterine cervix. In normal tissue p29 was present throughout the ectocervix during the menstrual cycle and virtually absent from the endocervix. In dysplastic cervical tissue there was a decreasing p29 content with increasing severity of the dysplasia, and very low levels were seen in the carcinomatous tissues.

Characterization and biological relevance of a 29-kDa, oestrogen receptor-related protein.

The properties of a monoclonal antibody (D5) that can immunoprecipitate human oestradiol receptor (ER) under some but not all conditions are described. The antibody recognises a 29-kDa serine phosphoprotein that is qualitatively and quantitatively related to ER but not other steroid receptors or binding proteins. p29 will not complex with untreated cytosol ER but, after ammonium sulphate, KCl, heat or phosphatase treatments, interaction occurs that can be detected by immunoprecipitation with D5; molybdate and GTP inhibit complex formation. In human endometrium, p29 is increased by oestrogen and decreased by progestins. IRMA and histochemical assays for p29 have been developed and applied to a large series of human breast tumours. Most, but not all ER+ tumours are p29+, whilst ER-tumours are rarely p29+ unless they are also PR+. p29 predicts for clinical response to hormone therapy. ER+ p29+ tumours have a higher response rate than the ER+ p29-tumours. We do not know if p29 is a previously undetected component of the oestradiol receptor machinery or whether it is a product of oestrogen action.

Histochemical studies with an estrogen receptor-related protein in human breast tumors.

The histochemical characteristics of a Mr 29,000 phosphoprotein related to estradiol receptor are described in a large series of human breast tumors. The antigen was detected with a monoclonal antibody (D5) raised against partially purified human myometrial estradiol receptor. An indirect immunoperoxidase method was used with methacarn-fixed, wax-embedded sections. Quantitation of staining and its reproducibility are described. Results with trucut biopsies agree with those obtained with larger tumor sections. Normal breast is infrequently positive. Histochemical staining is higher in invasive carcinoma than in normal breast with ductal carcinoma in situ adjacent to infiltrating tumors exhibiting intermediate values. Furthermore, most in situ carcinomas have a heterogeneous staining pattern. About 20% of invasive tumors also exhibit heterogeneity. No simple correlation is seen between staining and histological grade. There are more low-staining tumors in young (less than 50 yr old) patients than in older women. Staining correlates with levels of cytosol estradiol receptor but not cytosol progesterone receptor. However, cytosol estradiol receptor-negative, cytosol progesterone receptor-positive tumors tend to have positive Mr 29,000 phosphoprotein levels. Positive staining is associated with a higher response rate to hormone therapy (50%). None of the negative tumors responded to hormone treatment. With these patients, comparison of histochemical assay for Mr 29,000 phosphoprotein and [3H]estradiol binding assays indicated that the former was at least as good as the latter assay in predicting hormone response. About 20% of cytosol estradiol receptor-positive tumors have low Mr 29,000 phosphoprotein, and such tumors have poor response to hormone treatment.