AB Vector - StableHumanvsUnstableInsectHsp90

  Human steroid receptors form stable complexes with human Hsp90, but not with insect Hsp90

  Ligand-binding domains of steroid receptors were expressed in insect cells either supplemented or not supplemented with human molecular chaperones. Miniscule amounts of recombinant receptors were recovered if their expression in insect cells was not supplemented with human molecular chaperones and no detergents, high-salt buffers or chaotropic agents were applied to extract receptors from insoluble cell fraction (Fig.1). Such extractions were not applied as they typically result in dissociation of receptor/Hsp90 complexes. Both insect and human Hsp90 and Hsc70 formed receptor/Hsp90 complexes. However, receptor complexes with insect molecular chaperones were unstable and virtually all insect Hsp90 dissociated from the complexes after 3 days of storage at 4°C, whereas receptor/human Hsp90 complexes were stable. Similar observations were made for all steroid receptors (data not shown). This is not surprising as human steroid receptors are expected to interact better with human molecular chaperones rather than with insect molecular chaperones (details).

 





Fig. 1. Stability study of steroid receptor complexes formed with human or insect Hsp90.
Insect cells were co-infected with FoldHelper™-905c recombinant baculovirus expressing Hsp90, Hsp70, Hsp40, Hop and p23 human molecular chaperones and either recombinant baculovirus expressing GST-ERα-LBD (lanes 1,2) or GST-PR-LBD (lanes 5,6). Alternatively, insect cells were infected with either recombinant baculovirus expressing GST-ERα-LBD (lanes 3,4) or GST-PR LBD (lanes 7,8) without coexpressing them with human molecular chaperones.
Cells were destroyed in ice-cold K+ buffer (20 mM Na-P pH 8.0, 40 mM KCl, 20 mM Na2MoO4) using Dounce homogenizer. Soluble protein fractions were obtained after centrifugation in a microfuge at 14,000 r.p.m. at 4°C for 15 min and incubated for 4h with Glutathione Sepharose beads at 4°C. The beads were washed with the same buffer for 3 times and either stored for 3 days at 4°C to allow dissociation of the complexes, than washed additional 2 times and eluted (lanes 2,4,6,8), or washed additional 2 times immediately after the first 3 washes and eluted. Elution was performed at 4°C with 10 mM reduced glutathione dissolved in the same buffer. Proteins were identified using LS/MS/MS. Practically all insect Hsp90 and about a half of insect Hsc70 dissociated from the complexes (lanes 3,7), whereas receptor/human molecular chaperones (human Hsp90, human Hsc70 and human p23) ratios were not visibly affected (lanes 1,5).


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