Poly-amino acid repeats, especially long stretches of glutamine (Q), are common features of transcription factors and cell-signalling proteins and are prone to expansion, resulting in neurodegenerative diseases. The amino-terminal domain of the androgen receptor (AR-NTD) has a poly-Q repeat between 9 and 36 residues, which when it expands above 40 residues results in spinal bulbar muscular atrophy. We have used spectroscopy and biochemical analysis to investigate the structural consequences of an expanded repeat (Q45) or removal of the repeat (ΔQ) on the folding of the AR-NTD. Circular dichroism spectroscopy revealed that in aqueous solution, the AR-NTD has a relatively limited amount of stable secondary structure. Expansion of the poly-Q repeat resulted in a modest increase in α-helix structure, while deletion of the repeat resulted in a small loss of α-helix structure. These effects were more pronounced in the presence of the structure-promoting solvent trifluoroethanol or the natural osmolyte trimethylamine N-oxide. Fluorescence spectroscopy showed that the microenvironments of four tryptophan residues were also altered after the deletion of the Q stretch. Other structural changes were observed for the AR-NTDQ45 polypeptide after limited proteolysis; in addition, this polypeptide not only showed enhanced binding of the hydrophobic probe 8-anilinonaphthalene-1-sulphonic acid but was more sensitive to urea-induced unfolding. Taken together, these findings support the view that the presence and length of the poly-Q repeat modulate the folding and structure of the AR-NTD.
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Philippa Davies, Kate Watt, Sharon M Kelly, Caroline Clark, Nicholas C Price, and Iain J McEwan
SM MacKenzie, M Lai, CJ Clark, R Fraser, CE Gomez-Sanchez, Seckl JR, JM Connell, and E Davies
The central nervous system produces many of the enzymes responsible for corticosteroid synthesis. A model system to study the regulation of this local system would be valuable. Previously, we have shown that primary cultures of hippocampal neurons isolated from the fetal rat can perform the biochemical reactions associated with the enzymes 11beta-hydroxylase and aldosterone synthase. Here, we demonstrate directly that these enzymes are present within primary cultures of fetal rat hippocampal neurons.
A P Mee, L K Davenport, J A Hoyland, M Davies, and E B Mawer
ABSTRACT
The receptor for the active metabolite of vitamin D, 1,25(OH)2D3, known as the vitamin D receptor (VDR), belongs to the steroid hormone nuclear receptor superfamily. We have developed novel methods for detection of VDR mRNA and protein within a human promyelomonocytic cell line, HL-60.
Using the newly developed technique of in situ-reverse transcriptase-polymerase chain reaction (IS-RT-PCR), low levels of VDR mRNA could be amplified and demonstrated unequivocally within these cells, and also within a human kidney proximal tubule cell line, CL-8. Use of a novel immunogold cytochemical technique has allowed clear and sensitive detection of VDR protein expression within the HL-60 cells.
Further development of IS-RT-PCR has allowed us to apply this technique to tissue sections. We have shown clear amplification of VDR transcripts within sections of formalin-fixed paraffin-embedded human kidney and liver.
These techniques will be useful to localise specifically the VDR within cell types that contain low levels of mRNA and protein, and will permit further investigation of the role played by 1,25(OH)2D3 in cellular regulatory mechanisms.
J S Davies, J L Holter, D Knight, S M Beaucourt, D Murphy, D A Carter, and T Wells
Targeted overexpression of biologically active peptides represents a powerful approach to the functional dissection of neuroendocrine systems. However, the requirement to generate separate, biologically active and reporter molecules necessitates the use of internal ribosome entry site (IRES) technology, which often results in preferential translation of the second cistron. We report here a novel approach in which the proteolytic processing machinery of the regulated secretory pathway (RSP) has been exploited to generate multiple mature proteins from a monocistronic construct that encodes a single precursor. This was achieved by duplication of the pre-pro cleavage sites in pre-prosomatostatin cDNA. The duplicated site included 10 flanking amino acids on either side of the Gly-Ala cleavage position. This enabled the incorporation of a foreign protein-coding sequence (in this case, enhanced green fluorescent protein (eGFP)) between these sites. The pre-eGFP-prosomatostatin (PEPS) construct generated co-localized expression of fully processed eGFP and somatostatin to the RSP of transiently transfected AtT20 cells. This approach represents an advance upon bicistronic and other extant approaches to the targeting of multiple, biologically active proteins to neuroendocrine systems, and, importantly, permits the co-expression of fluorescent markers with biologically active neuropeptides. In this study, our demonstration of the fusion of the first 10 amino acids of the prosomatostatin sequence to the N-terminus of eGFP shows that this putative sorting sequence is sufficient to direct expression to the RSP.
M P A Davies, P A O’Neill, H Innes, D R Sibson, W Prime, C Holcombe, and C S Foster
This study has been performed to test the hypothesis that different oestrogen receptor beta (ERβ) splice variants may be important determinants of clinical parameters, including outcome, in post-menopausal women with breast cancer receiving adjuvant endocrine treatment but no chemotherapy. Splice variants ERβ1, ERβ2 and ERβ5 have been analysed by semi-quantitative RT-PCR in a cohort of 105 patients with primary breast cancer. Clinical correlates included age, grade, size, nodal status, ERα, progesterone receptor, Ki67, relapse-free survival (RFS) and overall survival (OS). Seventy per cent of cases were ERβ1 positive, 69% ERβ2 positive and 70% ERβ5 positive. Within the cohort, 47% were positive for all three variants while 10% were negative for all three. ERβ1 exhibited no discernible relationship with disease outcome. ERβ2 and ERβ5 expression was significantly associated with better RFS (P<0.005), and ERβ2 with better OS (P=0.0002). In multivariate analysis, ERβ2 (P=0.006), nodal status and the level of Ki67 expression were independent predictors for RFS while ERβ2 (P=0.0008) and Ki67 status were independent predictors for OS. In the ERα-positive cases, or in the subset of those receiving adjuvant tamoxifen, ERβ2 was significantly associated with good RFS (P<0.0005) and was the only independent marker of OS. We conclude that precise identification of splice variants of ERβ are more important assessors than is ERβ1 alone of the biological status of individual breast cancers, and hence in predicting their response to endocrine therapy.
L B Nicholson, H Vlase, P Graves, M Nilsson, J Molne, G C Huang, N G Morgenthaler, T F Davies, A M McGregor, and J P Banga
ABSTRACT
We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein. To increase the epitope specificity of the mAbs, two different strains of mice (H-2b and H-2d) were immunized. The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22–35 (A10 and All, both IgG2b from H-2b animals), amino acids 402–415 (A7, IgG2b from H-2b animals) and amino acids 147–228 (A9, IgG1 from H-2d animals). Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells. The binding of the mAbs A7, A10 and All was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule. None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and All, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function. The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.
