Hsp90-binding immunophilin FKBP51 forms complexes with hTERT enhancing telomerase activity

MARIANA LAGADARI; NADIA ZGAJNAR; SEBASTIAN SUSPERREGUY; GRACIELA PIWIEN PILIPUK; MARIO D. GALIGNIANA

Heraklion

Congreso; EMBO Conference on Molecular chaperones: From molecules to cells and misfolding diseases; 2015

EMBO

Telomeres gradually shorten with each cell division. When telomeres reach a critical length, cells cease dividing and undergo replicative senescence. Telomerase is an essential ribonucleoprotein that compensates the loss of telomeric DNA adding repeated sequences to the chromosome ends using its intrinsic RNA component as a template for DNA synthesis. The reverse transcriptase subunit of telomerase hTERT is the catalytic subunit, whereas the associated RNA component hTR serves as template. The minimal components necessary for telomerase assembly are hTERT, hTR, Hsp90, p23, Hsp70, Hsp40, and p60. While Hsp90 and p23 remain associated to the active telomerase, Hsp70 is bound to the inactive form. The onset of oxidative stress leads to the translocation of hTERT from the nuclei to mitochondria providing resistance to apoptosis. hTERT has non-telomere-related functions poorly characterized such as promotion of DNA repair, antagonism of cell apoptosis, alteration of the chromatin structure, and gene expression, all functions in favor of cell survival. The Hsp90-binding immunophilin FKBP51 was first described associated to steroid receptor heterocomplexes. Recently, we demonstrated that FKBP51 shows antiapoptotic action and is highly expressed in cancer cell lines and tumor tissues. Because hTERT is very active in those cell types where FKBP51 is also highly expressed, we analyzed whether this Hsp90-binding immunophilin forms complexes with hTERT and its biological role. The immunoprecipitation of hTERT from HeLa cell lysates yielded the coimmunoprecipitation of Hsp90 and FKBP51. This interaction was disrupted by geldanamycin and the overexpression of the TPR-domain peptide of the immunophilin. Confocal microscopy images for FKBP51 and hTERT show that hTERT is primarily nuclear and FKBP51 is ubiquitously distributed in mitochondria, cytosol and nuclei. Mask image analyses demonstrate that hTERT co-localizes with the nuclear fraction of FKBP51 in interphase, and with chromosomes in mitotic cells. Inasmuch as we have previously reported that FKBP51 translocates from mitochondria to the nucleus under conditions of oxidative stress exerting a protective role, HeLa cells were incubated with 0.25 mM H2O2, and after 60 min most, if not all FKBP51 population became nuclear colocalizing with nuclear hTERT. A fraction of hTERT population relocated to the cytoplasm, presumably in mitochondria. We investigated the potential interaction of FKBP51 with the telomeric region of DNA by chromatin immunoprecipitation assay (ChIP) and demonstrated that FKBP51 binds to chromatin. Specifically, we used primers to amplify the telomeric non-coding RNA (or actin as a non-specific control) in FKBP51- immunoprecipitated chromatin. In normal conditions, FKBP51 is able to bind to non-coding telomeric regions, perhaps repressing the expression of non-coding transcripts that permit the expression of certain factors whose activity should be abolished during harmful conditions to avoid major cell damage, as it is well documented in the literature. The potential inhibitory action is also implied by the role of FKBP51 on transcriptional regulation as we have already described for other systems such as steroid receptors, NF-êB, and AP-1 signalling cascades. Therefore, cells were exposed to oxidative stress with H2O2 as described above and a new ChIP assay was performed. FKBP51 was released from chromatin. Next we examined the role of FKBP51 on the hTERT enzymatic activity in a clone of HEK293 cells that stably overexpress hFKBP51, and in the HEK293T cell line transiently cotransfected with pCI-Neo-hFKBP51 and pCI-Neo-hEST2-HA-hTERT. Telomerase activity from cell lysates was measured and, in both systems, the increased levels of FKBP51 greatly induced hTERT enzymatic activity. This agrees with the overall role we assign to this immunophilin in cancer development and progression. In summary, we describe for the first time the association of the Hsp90- binding immunophilin FKBP51 to hTERT and the biological regulation of telomerase activity by this cochaperone.