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.

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.

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.

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.

Immunological probes for oestradiol receptors in human breast tumours.

Monoclonal antibodies have been prepared against a soluble oestradiol receptor (REC) preparation partially purified from human myometrium by oestradiol affinity chromatography. The antibodies were detected by their ability to immunoprecipitate receptor bound [125I] oestradiol. One of the antibodies (D5) has been studied in detail. It will only precipitate REC after activation by salt, heat, low pH or KCNS and will not react with nuclear RE. It will not react with androgen, progesterone or glucocorticoid receptors nor with sex hormone binding globulin; it will only combine with REC from human sources. D5 recognizes a cytoplasmic 29 kdalton protein (p29) that can be separated from both type I and II soluble oestradiol binding proteins. p29 can react with activated REC and is qualitatively and quantitatively related to REC. IRMA and histochemical methods have been developed for quantitating p29 and relating its amount to receptors in human breast tumours. With both methods, highly significant (P less than 0.001) correlations with REC but not RP have been obtained. Both methods indicate that many REC-RP+ tumours contain p29. The histochemical method detects marked cellular heterogeneity in some tumours. The function of p29 is not known. It is an REC-related antigen that may be a previously undetected component of the oestradiol receptor machinery.

Histochemical studies with a monoclonal antibody raised against a partially purified soluble estradiol receptor preparation from human myometrium.

A monoclonal antibody (D5) raised against affinity-purified cytosol estradiol receptor (REC) from human myometrium has been used to stain human tissues by means of an indirect immunoperoxidase method. Good staining was obtained with ethanol-, glutaraldehyde-, or Carnoy's-fixed material but not with formalin or Bouin's fixation. Cytoplasmic staining of human breast tumors exhibited a highly significant correlation (P less than 0.001) with REC assayed by conventional estradiol-binding assay provided that allowance was made for both staining intensity and cellularity of the tumor; no correlation existed with soluble progesterone receptor content. Both patient age and tumor differentiation influenced staining patterns in the same way as did REC content. Cultured REC-positive human breast tumor cell lines (MCF-7, ZR-75-1, and CA-2) showed positive staining as did cultured epithelium from human milk. Epithelia in normal breast and fibroadenoma exhibited variable staining that rarely reached the intensity seen in REC-positive tumor cells. The staining patterns of human normal endometrium, myometrium, fallopian tube, ectocervix, endocervix, and ovary and neoplastic endometrium and ovary are described. In every situation thus far examined only cytoplasmic staining has been observed.

Monoclonal antibodies against a component related to soluble estrogen receptor.

Mice were immunized with 1 to 3 micrograms of cytoplasmic estrogen receptor fragment purified from human myometrium by affinity chromatography. Two RE-antibody-secreting clones were detected from one fusion that were capable of precipitating cytosol RE. Monoclonal antibody D5 (subclass IgG1) reacts with an antigen that is related to RE from immunoprecipitation studies but which can be separated from the hormone binding unit. In the presence of anti-mouse serum, D5 precipitates labeled human cytoplasmic RE complexes from breast tumor, fibroid, myometrial, and endometrial preparations but does not react with nuclear RE from human endometrium or cytoplasmic RE from other species tested. Conversely, antibody C3 (Class IgM) precipitates human cytoplasmic RE and nuclear RE complexes as well as labeled cytoplasmic RE from rat and calf uterus and chick oviduct. Neither antibody reacts with progesterone receptor or androgen receptor from human breast tumor, SHBG from human plasma, or rat alpha-fetoprotein. With D5, steroid labeling of cytoplasmic RE at 25 degrees increased the RE immune complex precipitated. D5 precipitates molybdate-stabilized RE from myometrial cytosol when labeled at 25 degrees but not at 4 degrees. C5 precipitates molybdate-stabilized RE whether cytosol was steroid-labeled at 4 degrees or 25 degrees. For D5, optimal precipitation of RE from human breast tumor was observed when cytosol was steroid-labeled at 25 degrees in buffers of pH range 5 to 6. Immunochemical studies indicate that D5 is associated with a Mr 29,000 component in RE-positive cytosols. Electrofocusing and sucrose density gradient analysis confirmed that D5 antigen is a non-hormone-binding component related to cytosolic RE from breast tumor and myometrium.

Immunoradiometric studies with monoclonal antibody against a component related to human estrogen receptor.

A human specific monoclonal antibody (D5) raised against a Mr 36,000 cytosolic estrogen receptor component (RE) partially purified from human myometrium was used to develop a simple, rapid, and sensitive solid-phase immunoradiometric assay (IRMA) for the reactive antigen in tissue cytosols from breast tumors, myometrium, endometrium, and endometrial carcinomas. The IRMA did not detect antigen in RE-negative cytosols from human breast tumors and endometrial carcinomas. RE-positive cytosols from chick oviduct and calf and rat uteri failed to produce an IRMA response. A pilot study indicated a significant correlation (P less than 0.001) between D5 IRMA value and RE sites in breast tumors assayed by [3H]estradiol binding sites. The presence of D5 antigen was dependent on the presence of cytosolic RE but not progesterone receptor. RE-positive patients age 50 years and over demonstrated significantly higher D5 assay values than did patients under 50 years. The data suggest that the D5 antigen is a component of the estrogen receptor or coordinately regulated with the receptor in human cells and that the assay method may have clinical use.

Purification of oestradiol receptor from human uterus by affinity chromotrgraphy.

The oestrogen receptor from human myometrium has been extensively purified by affinity chromatography and isoelectric focusing. The latter step is necessary to remove contaminating sex-steroid-binding globulin. The unpurified 3.7-S cytoplasmic receptor has a molecular weight of 41,000, a Stokes radius of 27.0 A and a frictional ratio (f/f0) of 1.19; the KD (4 degrees C) for [3H]oestradiol-17 beta was 1.03 X 10(-10) M. After purification, the molecular weight was 30,000, the Stokes radius 23.6 A, frictional ratio 1.15 and isoelectric point 6.15.