Purification,characterization and crystallization in two crystal forms of bovine cyclophilin 40

The purification and crystallization of two different crystal forms of the two-domain protein bovine cyclophilin 40 is reported. Tetragonal crystals grown in methyl pentanediol belong to space group P4(2)22 with unit-cell parameters a = 94.5, c = 118.3 Angstrom. Long thin needles grown from PEG belong to space group C2 with unit-cell parameters a = 125.71, b = 47.3, c = 74.6 Angstrom, beta = 93.90 degrees. The N-terminal 170 amino acids have significant homology with the well characterized human cyclophilin A. The C-terminal domain is largely made up of three copies of the tetratricopeptide repeat motif thought to be involved in mediating protein-protein interactions. Cyclophilins are frequently found as domains in larger multidomain proteins. To date, only X-ray structures of single-domain cyclophilins have been reported, and this work provides the first example of the purification and crystallization of a larger protein containing a cyclophilin domain

Differential Impact of Tetratricopeptide Repeat Proteins on the Steroid Hormone Receptors

Tetratricopeptide repeat (TPR) motif containing co-chaperones of the chaperone Hsp90 are considered control modules that govern activity and specificity of this central folding platform. Steroid receptors are paradigm clients of Hsp90. The influence of some TPR proteins on selected receptors has been described, but a comprehensive analysis of the effects of TPR proteins on all steroid receptors has not been accomplished yet.We compared the influence of the TPR proteins FK506 binding proteins 51 and 52, protein phosphatase-5, C-terminus of Hsp70 interacting protein, cyclophillin 40, hepatitis-virus-B X-associated protein-2, and tetratricopeptide repeat protein-2 on all six steroid hormone receptors in a homogeneous mammalian cell system. To be able to assess each cofactor's effect on the transcriptional activity of on each steroid receptor we employed transient transfection in a reporter gene assay. In addition, we evaluated the interactions of the TPR proteins with the receptors and components of the Hsp90 chaperone heterocomplex by coimmunoprecipitation. In the functional assays, corticosteroid and progesterone receptors displayed the most sensitive and distinct reaction to the TPR proteins. Androgen receptor's activity was moderately impaired by most cofactors, whereas the Estrogen receptors' activity was impaired by most cofactors only to a minor degree. Second, interaction studies revealed that the strongly receptor-interacting co-chaperones were all among the inhibitory proteins. Intriguingly, the TPR-proteins also differentially co-precipitated the heterochaperone complex components Hsp90, Hsp70, and p23, pointing to differences in their modes of action.The results of this comprehensive study provide important insight into chaperoning of diverse client proteins via the combinatorial action of (co)-chaperones. The differential effects of the TPR proteins on steroid receptors bear on all physiological processes related to steroid hormone activity

Health economic modelling of overweight and obesity in Australian adolescents

Adolescence, defined as the phase of life from 10 years to 19 years of age, is a particularly vulnerable time for the development of overweight and obesity. This is a concern, as the earlier that excess weight is accumulated, the greater the associated morbidity, premature mortality and related costs in later life will be. As such, adolescence has been identified by policy-makers in Australia as an opportune time to intervene for overweight and obesity prevention and treatment. However, this is just one of many issues for decision-makers to consider within the constraints of finite resources. To make informed judgments for resource allocation, evidence that proposed interventions are cost-effective, or represent ‘value for money’ is required. While the costs of interventions for adolescent overweight and obesity are incurred up-front, their benefits are not fully realised until many years in the future, beyond what can be captured within a clinical trial. This makes it challenging to demonstrate their ‘value for money’. In these cases, modelling methods are required to estimate the costs and benefits of proposed interventions over longer-term and more policy-relevant timeframes. This thesis addresses this issue through the development of the Early Prevention of Obesity in CHildhood (EPOCH) Life-course model, a health economic model that can be applied to evaluate interventions for adolescent overweight and obesity in line with current best practice methodologies in Australia. Further, this thesis applies the EPOCH Life-course model to answer questions which will be of interest to current decision-makers in Australia. This includes demonstrating the economic benefits of a reduction in prevalence of overweight and obesity in Australian children and adolescents and the potential cost-effectiveness of e-health interventions as a possible strategy to achieve this

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

Biochemical and calmodulin binding properties of estrogen receptor binding cyclophilin expressed in Escherichia coli

Bovine estrogen receptor binding cyclophilin (ERBC), a cyclophilin component of the unactivated estrogen receptor, has been efficiently expressed in Escherichia coli as a fusion with glutathione S-transferase (GST) and purified by single-step chromatography on glutathione-agarose. Thrombin cleavage from GST allowed the isolation of purified, recombinant ERBC. The fusion protein, GST-ERBC, and recombinant ERBC were both characterised for peptidyl prolyl cis-trans isomerase activity. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate, GST-ERBC demonstrated a k/K value of 5.1 × 10 M s at 5°C. The isomerase activity was inhibited by cyclosporin A with an IC value of 1030 nM. These values indicate that ERBC has a decreased catalytic efficiency and sensitivity to cyclosporin A relative to human cyclophilin. Retention of the GST-ERBC fusion protein on calmodulin-agarose in the presence of Ca and subsequent elution with EGTA has provided evidence that ERBC is a calmodulin-binding protein

Nuclear-accumulation kinetics of p9CksHs1 and p9CksHs2 in live plant cells correlate with immunochemical characteristics

The two human homologues of the fission yeast cell cycle protein p13(suc1) displayed structural characteristics consistent with their existing in solution as differently folded monomers despite 81% identity with respect to their primary structures and both being capable of fulfilling the functions of their homologues in fission and budding yeasts. Carboxyfluorescein-labelled p(9CksHs1) and p(9CksH2) retained their native structures. When microinjected into live stamen hair cells of Tradescantia virginiana, the labelled proteins accumulated in the nuclei of the cells. Markedly different nuclear-accumulation kinetics indicated that the human proteins interact differently with other cellular constituents, which supports the proposition that they may have different roles in cellular regulation