AB Vector - Interaction of Steroid Receptors

  Interaction of steroid receptors with homologous and heterologous molecular chaperones
Misfolding of recombinant Hsp90-dependent proteins in heterologous expression systems appears to be in part due to weaker interaction of heterologous molecular chaperones with recombinant proteins of interest. Evolutionary, Hsp90 function is domain and kingdom specific and, to a degree, phylum-specific. For instance, E.coli Hsp90 cannot facilitate native folding of steroid receptors. Therefore, human steroid receptors are inactive if expressed in E.coli (Jaglaguier et al., J Steroid Biochem Mol Biol., 57: 43-50, 1996). Plant molecular chaperones are also incapable of supporting mammalian steroid receptor activity. For instance, glucocorticoid receptor (GR) translated in wheat germ extract was not associated with wheat Hsp90 and did not bind steroid with high affinity, whereas GR translated in rabbit reticulocyte lysate was in complex with rabbit Hsp90 and attained high affinity steroid binding conformation (Pratt et al., Nihon Naibunpi Gakkai Zasshi, 66:1185-97, 1990).

  Most human steroid receptors exhibit ligand-binding activity if expressed in insect cells and it is the system of choice for producing these receptors for drug screening. However, even insect Hsp90 does not interact with human steroid receptors as well as human or even avian Hsp90. Therefore ligand binding is not as competent. For example, when human GR, chicken Hsp90 and insect Hsp90 were co-expressed in insect cells, GR ligand will preferentially bind to GR/chicken Hsp90 complexes versus the GR/insect Hsp90 complexes (Cadepond et al., Proc. Natl. Acad. Sci. USA, 90:10434-8, 1993). Likewise, we observed weak interaction of human steroid receptors with indigenous insect Hsp90 as compared to the receptors interaction with recombinant human Hsp90 expressed in insect cells (details). Amount of soluble receptors that was recovered was miniscule if no human molecular chaperones were coexpressed with the receptors. Without human chaperones the total receptor production was not drastically affected, but receptors were largely misfolded and produced in a form of protein aggregates; and it was possible to recover significant amounts of the receptors only by disrupting the aggregates. However in our experience this yielded only apo-Hsp90 receptors.