ISBN 9783868538434

978-3-86853-843-4, Reihe Biochemie

Andreas M. Gaiser
Studies on the molecular chaperone Hsp90 and its regulation by co-chaperones in Caenorhabditis elegans

223 Seiten, Dissertation Technische Universität München (2010), Softcover, A5

Zusammenfassung / Abstract

Despite the many cellular processes which rely on Hsp90 as a molecular chaperone, little is known about Hsp90 functions in a multi‐cellular organism. Bacterial homologs of Hsp90 are not essential, although knockout results in growth defects especially under stress conditions. In yeast, however, Hsp90 seems to carry out essential functions, as the deletion of both copies leads to growth arrest. Moreover, studies in Drosophila and mammals revealed that the absence of functional Hsp90 results in severe damages during development. It seems that with increasing developmental complexity, Hsp90 loss of functions become more severe, potentially due to the many client proteins which are known to exist in eukaryotes. The purpose of this work was to introduce C. elegans as a model organism for the study of Hsp90 functions in vitro and in vivo. C. elegans has the advantage over S. cerevisae that it contains homologs for most of the mammalian Hsp90 substrate proteins in its genome, but yet is simple enough to be studied in great detail.

The protein Hop/Sti1 has been identified as an Hsp70/Hsp90‐organizing protein during the activation of SHRs in mammalian cells. Hop/Sti1 was found to bind to an early complex of SHR, Hsp70 and Hsp40 and to ensure substrate transfer from Hsp70 to Hsp90 by connecting the two chaperones in a ternary complex. Interestingly, the nematode homolog STI1 lacks the Hsp70 interacting TPR1 domain, while the TPR domain required for the interaction with Hsp90 is fully conserved. Therefore, the aim was to test whether the described function of STI1 as an adapter protein is also conserved in the nematode system. STI1 binds strongly to Hsp90 and inhibits its ATPase activity, which is similar to the yeast homolog, but in contrast to the human system in which no ATPase inhibition of human Hsp90 by Hop has been found. In contrast, only a very weak interaction between STI1 and Hsc70 could be detected, even in the absence of the described binding site. However, no influence on the ATPase activity was observable. Surface plasmon resonance competition experiments revealed a complete disruption of Hsc70 binding to STI1 in the presence of Hsp90. The competitive binding of Hsc70 and Hsp90 to STI1 implies the presence of overlapping binding sites in the absence of the TPR1 domain, which would exclude the formation of ternary complexes in nematodes. RNA interference studies revealed an involvement of STI1 in gonad formation, similar to the defects observed for an Hsp90 knockdown. hsf‐ 1(sy441)/R09E12.3(RNAi) worms remained sterile with a penetrance of 100% and exhibited a distorted gonad structure. In addition, the spermathecae, which are required for the fertilization of oocytes, were less developed. This implies that STI1 and Hsp90 may cooperate and participate in processes that are required for the development of fertilitiy.