The molecular chaperone Hsp90 is required for the function of multiple eukaryotic growth regulatory signaling proteins and in the cellular response to stress. The protein sequences of Hsp90s are highly conserved across species. Hsp90 protein is the target of the antitumor drug geldanamycin (GA) - a derivative of which is currently in clinical trials for cancer treatment. C. elegans possesses a single ortholog of Hsp90 called Daf21, whose protein sequence is 74% and 76% identical to human Hsp90 alpha and beta respectively. Despite this conservation in protein sequence, DAF21 is the only Hsp90 examined to date that does not bind GA. The first goal of our research is to determine the 3-dimensional structure of DAF21 in order to identify the structural features that confer this resistance to GA. Our second goal is to test the function of human Hsp90 in C. elegans
daf21 mutants (RNAi and deletion) in order to develop a C. elegans -based system for drug assays and screening. For the structural studies, we have expressed His-tagged versions of the N-terminal domain (shown to be involved in GA-binding in other species) as well as the full-length DAF21 in E.coli , and purified the proteins to homogeneity using Ni-column affinity chromatography followed by ion-exchange chromatography. The structure of these proteins will be determined using X-ray crystallography and NMR-spectroscopy. For the functional studies, we first performed RNAi of
daf21 using the feeding method. We observed a range of phenotypes - slow growth, embryonic lethality, vulval defects, dauer-like appearance and sterility. We made constructs comprising cDNAs of the human Hsp90 alpha and
daf-21 under the control of the
daf-21 promoter. We have created transgenic worms expressing these plasmids using microparticle bombardment. We are currently analyzing these transgenic worms for complementation of the RNAi phenotypes.