HSP90-BINDING IMMUNOPHILIN FKBP51 FORMS COMPLEXES WITH hTERT ENHANCING TELOMERASE ACTIVITY

LAGADARI M, ZGAJNAR NR, SUSPERREGUY S, PIWIEN PILIPUK, GALIGNIANA MD

Congreso; EMBO Meeting on Molecular Chaperones: From Molecules to Cells and Misfolding Diseases; 2015

EMBO

Telomeresgradually shorten with each cell division. When telomeres reach a critical length,cells cease dividing and undergo replicative senescence. Telomerase is anessential ribonucleoprotein that compensates the loss of telomeric DNA addingrepeated sequences to the chromosome ends using its intrinsic RNA component asa template for DNA synthesis. The reverse transcriptase subunitof telomerase hTERT is the catalytic subunit, whereas the associated RNAcomponent hTR serves as template. The minimal components necessary for telomeraseassembly are hTERT, hTR, Hsp90, p23, Hsp70, Hsp40, and p60. While Hsp90 and p23remain associated to the active telomerase, Hsp70 is bound to the inactiveform. The onset of oxidative stress leads to the translocation of hTERT fromthe nuclei to mitochondria providing resistance to apoptosis. hTERT hasnon-telomere-related functions poorly characterized such as promotion of DNArepair, antagonism of cell apoptosis, alteration of the chromatin structure,and gene expression, all functions in favor of cell survival. The Hsp90-bindingimmunophilin FKBP51 was first described associated to steroid receptorheterocomplexes. Recently, we demonstrated that FKBP51 shows antiapoptoticaction and is highly expressed in cancer cell lines and tumor tissues. BecausehTERT is very active in those cell types where FKBP51 is also highly expressed,we analyzed whether this Hsp90-binding immunophilin forms complexes with hTERT andits biological role. The immunoprecipitation of hTERT from HeLa cell lysatesyielded the co-immunoprecipitation of Hsp90 and FKBP51. This interaction wasdisrupted by geldanamycin and the overexpression of the TPR-domain peptide ofthe immunophilin. Confocal microscopy images for FKBP51 and hTERT showthat hTERT is primarily nuclear and FKBP51 is ubiquitously distributed inmitochondria, cytosol and nuclei. Mask image analyses demonstrate that hTERTco-localizes with the nuclear fraction of FKBP51 in interphase, and with chromosomesin mitotic cells. Inasmuch as we have previously reportedthat FKBP51translocates from mitochondria to the nucleus under conditions of oxidativestress 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 colocalizingwith nuclear hTERT. A fraction of hTERT population relocated to the cytoplasm,presumably in mitochondria. We investigated the potential interaction of FKBP51 withthe telomeric region of DNA by chromatin immunoprecipitation assay (ChIP) anddemonstrated that FKBP51 binds to chromatin. Specifically, we used primers to amplify the telomeric non-coding RNA (or actin as a non-specific control) inFKBP51-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 theexpression of certain factors whose activity should be abolished during harmfulconditions to avoid major cell damage, as it is well documented in theliterature. The potential inhibitory action is also implied by the role ofFKBP51 on transcriptional regulation as we have already described for othersystems such as steroid receptors, NF-kB, and AP-1 signalling cascades. Therefore, cells were exposed tooxidative stress with H2O2as described above and a new ChIP assay was performed. FKBP51 was released fromchromatin. Next we examined the role of FKBP51 on the hTERT enzymatic activity in a clone of HEK293 cells that stablyoverexpress hFKBP51, and in the HEK293T cell line transiently co-transfected 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 FKBP51greatly induced hTERT enzymatic activity. This agrees with the overall role we assign to thisimmunophilin in cancer development and progression. In summary, we describe forthe first time the association of the Hsp90-binding immunophilin FKBP51 tohTERT and the biological regulation of telomerase activity by this cochaperone.