The Hsp90 Molecular Chaperone Modulates Multiple Telomerase Activities

Abstract

The Hsp90 molecular chaperone is a highly abundant eukaryotic molecular chaperone. While it is understood that Hsp90 modulates a significant number of proteins, the mechanistic contributions made by Hsp90 to a client protein typically are not well understood. Here we investigate the yeast Hsp90 regulatory roles with telomerase. Telomerase lengthens chromosome termini by specifically associating with single-stranded telomeric DNA and appending nucleotides by reverse transcription. We have found that the yeast Hsp90 homolog Hsp82p promotes both telomerase DNA binding and nucleotide addition properties. By isolating telomerase from different allelic backgrounds we observed distinct defects. For example, in an hsp82 T101I strain telomerase displayed decreased nucleotide processivity, whereas both DNA binding and extension activities were lowered in a G170D background. The decline in telomerase DNA binding correlated with a loss of Hsp82p association. No matter the defect, telomerase activity was recovered upon Hsp82p addition. Importantly, telomere length and telomerase telomere occupancy was yeast Hsp90 dependent. Taken together, our results indicate that Hsp82p promotes telomerase DNA association and facilitates DNA extension once telomerase is engaged with the DNA.The Hsp90 molecular chaperone is a highly conserved and abundant protein that has evolved into an essential eukaryotic protein (4, 13, 41). Given recent proteomic and genetic screens, Hsp90 has a role in a multitude of normal cellular functions and is involved in a number of diseases ranging from conformational protein folding problems to cancer (31). Accelerating the interest in Hsp90 is its developing use as a therapeutic target for diverse diseases (27,42). Presumably, the broad therapeutic spectrum results from Hsp90's role in maintaining the conformation, stability, and activity of many key cellular proteins that includes intracellular hormone receptors, cRaf, Her2, Akt, Cdk4, p53, and telomerase. Despite its apparent central role in the eukaryotic molecular chaperone system and disease relevance, its mechanistic role in client protein regulation is not well understood. Here we investigate the yeast Hsp90 contributions to telomerase activities.Telomerase maintains genomic integrity, in part, by preserving chromosome length after DNA replication (5, 34). Since conventional DNA polymerases require priming events to initiate synthesis, the extreme terminus of each lagging strand cannot be completed-commonly referred to as the end replication problem (39). In the absence of a compensatory process, this limitation would lead to chromosome erosion with each round of replication. Almost all eukaryotes circumvent this problem by adding a tandem array of simple sequence repeats to each terminus that buffers against the loss. Depending upon the organism, telomerase increases chromosome ends between a few hundred to a few thousand nucleotides to create the telomeric DNA end (34). Perhaps unexpectedly, it was realized that the length of each telomere is not ad...

Inhibition of HSP90‐dependent telomerase activity in amyloid β‐induced apoptosis of cerebral endothelial cells

“…HSP90 has been shown as functionally being a critical factor for telomerase activity in vivo and in vitro (Keppler et al, 2006;Toogun et al, 2008). Mizuno et al (2007) telomerase complex with HSP90 is sensitive to HSP90 inhibitor GA and GA-inhibited telomerase activity via stimulated proteasome-mediated degradation of TERT, they are prevented by proteasome inhibitors, MG132.…”

Structure and Functional Studies of the CS Domain of the Essential H/ACA Ribonucleoparticle Assembly Protein SHQ1

“…Both Hsp90 and one of its co-chaperone proteins p23, which is structurally similar to the CS domain (hence the CS domain is also called as p23-like domain), have also been shown to bind to hTERT and influence its assembly process with hTR (54,55). Hsp82p was also reported to promote both the DNA binding and nucleotide extension properties of telomerase in yeast (56 15 N HSQC spectra of Shq1p CS even with a 3-fold excess of Hsp82p, indicating that there is no interaction between the two proteins under the NMR conditions (Fig. 7).…”

Stimulation of Yeast Telomerase Activity by the Ever Shorter Telomere 3 (Est3) Subunit Is Dependent on Direct Interaction with the Catalytic Protein Est2

“…Biotin-labeled yeast telomeric primers were bound to streptavidin beads and incubated in the presence of radiolabeled nucleotides and wild-type telomerase extract that was partially purified over DEAE and Mono Q resins (5,29). As previously demonstrated, yeast telomerase adds seven nucleotides (5Ј-GGTGTGG) to the end of the primer and then terminates elongation.…”