Mutational analysis of the hsp70-interacting protein Hip.

The hsp70-interacting protein Hip participates in the assembly pathway for progesterone receptor complexes. During assembly, Hip appears at early assembly stages in a transient manner that parallels hsp70 interactions. In this study, a cDNA for human Hip was used to develop various mutant Hip forms in the initial mapping of functions to particular Hip structural elements. Hip regions targeted for deletion and/or truncation included the C-terminal region (which has some limited homology with Saccharomyces cerevisiae Sti1 and its vertebrate homolog p60), a glycine-glycine-methionine-proline (GGMP) tandem repeat, and a tetratricopeptide repeat (TPR). Binding of Hip to hsp70's ATPase domain was lost with deletions from the TPR and from an adjoining highly charged region; correspondingly, these Hip mutant forms were not recovered in receptor complexes. Truncation of Hip's Sti1-related C terminus resulted in Hip binding to hsp70 in a manner suggestive of a misfolded peptide substrate; this hsp70 binding was localized to the GGMP tandem repeat. Mutants lacking either the C terminus or the GGMP tandem repeat were still recovered in receptor complexes. Truncations from Hip's N terminus resulted in an apparent loss of Hip homo-oligomerization, but these mutants retained association with hsp70 and were recovered in receptor complexes. This mutational analysis indicates that Hip's TPR is required for binding of Hip with hsp70's ATPase domain. In addition, some data suggest that hsp70's peptide-binding domain may alternately or concomitantly bind to Hip's GGMP repeat in a manner regulated by Sti1-related sequences.     

Progesterone receptor structure and function altered by geldanamycin, an hsp90-binding agent.

The assembly of progesterone receptor (PR) heterocomplexes in vitro involves at least eight components of the molecular chaperone machinery, and as earlier reports have shown, these proteins exhibit complex, dynamic, but ordered, interactions with one another and PR. Using the selective hsp90 binding agent geldanamycin (GA), we have found that PR assembly in vitro can be arrested at a previously observed intermediate assembly step. Like mature PR complexes, the intermediate complexes contain hsp90, but they differ from mature complexes by the presence of hsp70, p60, and p48 and the absence of immunophilins and p23. Arrest of PR assembly is likely due to GA's ability to directly block binding of p23 to hsp90. An important functional consequence of GA-mediated assembly arrest in vitro is the inability of the resulting PR complexes to bind progesterone, despite the presence of hsp90 in the receptor complexes. The biological significance of the in vitro observations is demonstrated by GA's ability to (i) rapidly block PR's hormone binding capacity in intact cells and (ii) alter the composition of COS cell PR complexes in a manner similar to that observed during in vitro reconstitutions. An updated model for the cyclic assembly pathway of PR complexes that incorporates the present findings with earlier results is presented.     

Molecular cloning of human FKBP51 and comparisons of immunophilin interactions with Hsp90 and progesterone receptor.

A cDNA for human FKBP51 has been cloned and sequenced, and protein products have been expressed in both in vitro and bacterial systems. The deduced amino acid sequence for human FKBP51 is 90% identical to sequences of recently described murine proteins and is 55% identical to the sequence of human FKBP52. Human FKBP51 mRNA is expressed in a wide range of tissues, and the protein has peptidylprolyl isomerase activity that is inhibited by FK506 but not cyclosporine. FKBP51 is the same as a previously described progesterone receptor-associated immunophilin that, similar to FKBP52 and cyclophilin 40, is an Hsp90-binding protein and appears in functionally mature steroid receptor complexes along with Hsp90 and p23. Each of the three receptor-associated immunophilins displays interactions with progesterone receptor that are more dynamic than Hsp90-receptor interactions. Whereas FKBP52 and FKBP51 compete about equally well for binding to Hsp90 in a purified system, FKBP51 accumulates preferentially in progesterone receptor complexes assembled in a cell-free system. This observation provides a precedent for differential interactions between Hsp90-associated immunophilins and target proteins such as steroid receptors.     

The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo

Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52.     

Physiological role for the cochaperone FKBP52 in androgen receptor signaling

Molecular chaperones mediate multiple aspects of steroid receptor function, but the physiological importance of most receptor-associated cochaperones has not been determined. To help fill this gap, we targeted for disruption the mouse gene for the 52-kDa FK506 binding protein, FKBP52, a 90-kDa heat shock protein (Hsp90)-binding immunophilin found in steroid receptor complexes. A mouse line lacking FKBP52 (52KO) was generated and characterized. Male 52KO mice have several defects in reproductive tissues consistent with androgen insensitivity; among these defects are ambiguous external genitalia and dysgenic prostate. FKBP52 and androgen receptor (AR) are coexpressed in prostate epithelial cells of wild-type mice. However, FKBP52 and AR are similarly coexpressed in testis even though testis morphology and spermatogenesis in 52KO males are usually normal. Molecular studies confirm that FKBP52 is a component of AR complexes, and cellular studies in yeast and human cell models demonstrate that FKBP52 can enhance AR-mediated transactivation. AR enhancement requires FKBP52 peptidylprolyl isomerase activity as well as Hsp90-binding ability, and enhancement probably relates to an affect of FKBP52 on AR-folding pathways. In the presence of FKBP52, but not other cochaperones, the function of a minimally active AR point mutant can be dramatically restored. We conclude that FKBP52 is an AR folding factor that has critically important physiological roles in some male reproductive tissues.     

Detection of venom by enzyme linked immunosorbent assay (ELISA) in patients bitten by snakes in Thailand.

The ability of an enzyme linked immunosorbent assay (ELISA) to detect venom was evaluated in 251 patients bitten by four of the commonest poisonous snakes in Thailand. Serum was tested only from patients who brought the snakes that had bitten them. About one third of all bitten patients had detectable venom antigenaemia, though a smaller proportion were symptomatic. Serum venom concentrations on admission correlated with the severity of clinical manifestations. The test was sensitive and specific even for specimens that had been collected and stored under suboptimal conditions. The technique is suitable for forensic use in cases of suspected snakebite. The combination of snake identification and venom antigen detection should be a more reliable means of studying the epidemiology of snakebite than the measurement of venom antibodies in a population.     

Outbreak ofTinea capitis caused by Microsporum ferrugineum in Thailand

There was an outbreak ofTinea capitis at the Pak-kred Home for Mentally and Physically Handicapped Babies, Bangkok, Thailand in 1993. One hundred and thirty-eight cases were diagnosed as tinea capitis based on clinical signs and positive laboratory investigations. The results of Wood's light examination, KOH preparation and fungal culture were positive in 89.9, 75.9 and 27.4% respectively. The non-inflammatory form had a higher rate of positive KOH and culture than in the inflammatory form.Microsporum ferrugineum was the major pathogen (66.7%) and most of its infections (80.4%) caused a non-inflammatory type of tinea capitis. Griseofulvin, in a dosage of 10–15 mg/kg/day and selenium sulfide shampoos, yielded an 84.8% cure rate within 14.9 weeks. No recurrence or obvious adverse reactions were observed.