Thomas Ratajczak

Cyclophilin 40 (CyP-40), Mapping of Its hsp90 Binding Domain and Evidence That FKBP52 Competes with CyP-40 for hsp90 Binding

The structurally related immunophilins cyclophilin 40 (CyP-40) and FKBP52 have been identified as components of the unactivated estrogen receptor. Both immunophilins have a similar molecular architecture that includes a C-terminal segment with a tetratricopeptide repeat (TPR) domain predicted to mediate protein interaction. hsp90 is a common cellular target for CyP-40 and FKBP52. Deletion mutants of CyP-40 fused to glutathione S-transferase were immobilized on glutathione-agarose and then used in a rapid hsp90 retention assay to define regions of the CyP-40 C terminus that are important for hsp90 binding. Our evidence suggests that the TPR domain is not sufficient for stable association of CyP-40 with hsp90 and requires the participation of flanking acidic and basic residues clustered at the N- and C-terminal ends, respectively. Both microdomains are characterized by α-helical structures with segregated hydrophobic and charged residues. Corresponding regions were identified in FKBP52. By preincubating myometrial cytosol with lysates containing bacterially expressed FKBP52, we have shown that FKBP52 competes with CyP-40 for hsp90 binding. Our results raise the possibility of a mutually exclusive association of CyP-40 and FKBP52 with hsp90. This would lead to separate immunophilin-hsp90-receptor complexes and place the estrogen receptor under the control of distinct immunophilin signaling pathways.

The common tetratricopeptide repeat acceptor site for steroid receptor-associated immunophilins and Hop is located in the dimerization domain of hsp90

Structurally related tetratricopeptide repeat motifs in steroid receptor-associated immunophilins and the STI1 homolog, Hop, mediate the interaction with a common cellular target, hsp90. We have identified the binding domain in hsp90 for cyclophilin 40 (CyP40) using a two-hybrid system screen of a mouse cDNA library. All isolated clones encoded the intact carboxyl terminus of hsp90 and overlapped with a common region corresponding to amino acids 558–724 of murine hsp84. The interaction was confirmed in vitro with bacterially expressed CyP40 and deletion mutants of hsp90β and was delineated further to a 124-residue COOH-terminal segment of hsp90. Deletion of the conserved MEEVD sequence at the extreme carboxyl terminus of hsp90 precludes interaction with CyP40, signifying an important role for this motif in hsp90 function. We show that CyP40 and Hop display similar interaction profiles with hsp90 truncation mutants and present evidence for the direct competition of Hop and FK506-binding protein 52 with CyP40 for binding to the hsp90 COOH-terminal region. Our results are consistent with a common tetratricopeptide repeat interaction site for Hop and steroid receptor-associated immunophilins within a discrete COOH-terminal domain of hsp90. This region of hsp90 mediates ATP-independent chaperone activity, overlaps the hsp90 dimerization domain, and includes structural elements important for steroid receptor interaction.

Allelic loss of cyclophilin 40, an estrogen receptor-associated immunophilin, in breast carcinomas.

Purpose: Cyclophilin 40 (CyP40) is an estrogen receptor-associated protein which appears to modify receptor function. The aim of this study was to determine the extent of allelic loss at the CyP40 locus in a panel of breast carcinomas using a newly characterized microsatellite marker located upstream of the CyP40 gene and then to correlate this with losses at chromosomal sites for cancer-associated genes. Methods: Allelic loss at CyP40 was determined from patients matched tumor and normal breast tissue using Genescan 672 software analysis of fluorescently labeled, PAGE-separated PCR products incorporating the marker. For each patient, allelic loss at CyP40 was then assessed and compared with losses at markers for various cancer-associated genes. Results: Allelic loss was detected in 30% of breast carcinomas from patients heterozygous for the CyP40 marker. All carcinomas demonstrating allelic loss were grade II or III invasive ductal carcinomas and generally showed multiple losses at other sites near known cancer-associated genes. Conclusions: The polymorphic marker which we characterized was useful in determining allelic loss at the CyP40 locus in breast cancer patients and when applied in these studies in conjunction with various cancer-associated gene markers, suggests that deletions in the region of the CyP40 gene might be a late event in breast tumor progression.

Regulation of the 1b isoform of the plasma membrane calcium pump by 1,25-dihydroxyvitamin D3 in rat osteoblast-like cells.

The first isogene of the plasma membrane calcium pump (PMCA1) is expressed on the apical plasma membrane of osteoblasts, but its regulation by 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] has not been studied in this cell type. We studied 1,25(OH)2D3 effects on PMCA1 function, protein, messenger RNA (mRNA), and isoform expression in osteoblasts. Of seven rat and human immortalized osteoblast‐like cell lines studied, PMCA1 mRNA expression was confirmed in all. Only ROS 17/2.8 cells expressed measurable PMCA1 protein by Western analysis. Immunocytochemistry indicated that PMCA1 was expressed predominantly on the plasma membrane of ROS 17/2.8 cells. The 1,25(OH)2D3 but not 24,25‐dihydroxyvitamin D3 [24,25(OH)2D3] treatment of confluent ROS 17/2.8 cells resulted in an approximate 3‐ to 5‐fold dose‐dependent increase in PMCA1 expression of message and protein as assessed by Western and Northern analysis and vesicular45Ca uptake of membrane vesicles. 1,25(OH)2D3 had no effect on PMCA1 posttranscriptional splicing. The 1b isoform of PMCA was expressed under all experimental conditions. 1,25(OH)2D3 favored increased expression of the 5.5 kilobases (kb) over the 7.5‐kb PMCA1b transcript, with a 2‐fold proportional increase in the smaller transcript relative to the larger transcript evident at the highest dose of 1,25(OH)2D3 studied. The resultant proportional increase in the smaller 5.5‐kb transcript may increase mRNA stability and account for the increase in PMCA1b protein and function with 1,25(OH)2D3. These data provide evidence for the role of 1,25(OH)2D3 and PMCA1b in the regulation of calcium transport in bone cells.

Cyclophilin 40 (PPID) gene Map position 4q31.3

The structurally related immunophilins cyclophilin 40 (CyP40) and FKBP52 have been identified as components of assembled steroid receptors [1] and may modulate receptor biological activity and that of other signalling proteins [2] through a common interaction with hsp90 [3]. Genomic fragments corresponding to the 5 flanking region (4.5 kb) and to the first intron (1.7 kb) of the human CyP40 gene (PPID) [4] were generated by PCRbased methodology using CyP40 gene-specific primers (T. Ratajczak et al, unpublished). CyP40 probes derived from these fragments were used together in fluorescence in situ hybridization studies conducted as described previously [5]. Chromosomes were stained before analysis with both propidium iodide (as counterstain) and DAPI (for chromosome identification). Twenty-five metaphases from a normal male all showed specific labelling of 4q31.3 on all four chromatids in 5 cells, three chromatids in 2 cells, two chromatids in 6 cells and one chromatid in 12 cells. Nine non-specific background dots were observed. A similar result was obtained from hybridization of the probes to 15 metaphases from a second normal male, confirming the 4q31.3 position. Identification of chromosome 4 was assisted by the use of a cosmid probe B31, which hybridizes to the 4pter region. These data confirm and further define the previous localization of the human CyP40 gene to chromosome 4 by PCR analysis of a human–rodent somatic hybrid cell panel [4]. This work was supported by the National Health and Medical Research Council of Australia, Cancer Foundation of Western Australia and The Kathleen Cuningham Foundation. 1. Ratajczak T et al. (1993) J Biol Chem 268: 13187–13192 2. Kimura Y et al. (1995) Science 268: 1362–1365. 3. Ratajczak T & Carrello A (1996) J Biol Chem 271: 2961–2965. 4. Yokoi H et al. (1996) Genomics 35: 448–455. 5. Callen D F et al. (1990) Ann Genet 33: 219–221.

The Changing Presentation of Paget’s Disease of Bone in Australia, A High Prevalence Region

Studies from several countries suggest that the incidence of Paget’s disease of bone (PDB) and the severity of newly diagnosed cases are declining. The aim of this study was to examine secular changes in clinical presentation of PDB in Australia, which historically had the highest prevalence outside the United Kingdom. The participants were 293 patients (61% male) diagnosed between 1956 and 2013 with details recorded in the database of the Paget’s Disease Research Group of Western Australia. The mean age at diagnosis was 62xa0years (range 28–90); 26% of participants had a family history of PDB and 11% had Sequestosome 1 (SQSTM1) mutations. After adjustment for covariates (SQSTM1 mutation status, family history, country of birth, smoking and dog exposure), there was a significant positive relationship between year of diagnosis and age at diagnosis (Pxa0<xa00.001) and significant negative relationships between year of diagnosis and both pre-treatment total plasma alkaline phosphatase activity (ALP) and number of involved bones (Pxa0<xa00.001 for each). Patients with SQSTM1 mutations had more extensive disease (Pxa0<xa00.001) and higher pre-treatment ALP (Pxa0=xa00.013). In subgroup analyses, relationships between year of diagnosis and each of age at diagnosis, number of involved bones and ALP were similar in patients with sporadic or familial disease, and in patients with and without SQSTM1 mutations. We conclude that the severity of PDB in Western Australia has declined over recent decades. This is likely to reflect altered exposure to one or more environmental agents involved in pathogenesis.

Familial Hypocalciuric Hypercalcaemia in Australia

1. Stuckey BGA, Kent GN, Gutteridge DH, Pullan PT, Price RI, Bhagat C (1987) Fasting calcium excretion and parathyroid hormone together distinguish familial hypocalciuric hypercalcaemia from primary hyperparathyroidism. Clin Endocrinol 27:525–533. 2. Stuckey BGA, Gutteridge DH, Kent GN, Reed WD (1990) Familial hypocalciuric hypercalcaemia and pancreatitis: no causal link proven. Aust NZ J Med 20:718– 719. 3. Ward BK, Stuckey BGA, Gutteridge DH, Laing NG, Pullan PT, Ratajczak T (1997) A novel mutation (L174R) in the Ca-sensing receptor gene associated with familial hypocalciuric hypercalcaemia. Human Mutation 10:233–235.