Human Telomerase Exists in Two Distinct Active Complexes In Vivo

Abstract

Telomerase, a stable complex of telomerase reverse transcriptase (TERT) and template RNA (TERC), is responsible for telomere maintenance. During purification trials of recombinant human telomerase of the two components reconstituted in insect cells, we identified two complexes of human telomerase of molecular masses 680 and 380 kDa, both of which retain telomerase activity in vitro. We show here that the former complex does not include Hsp90 (heat shock protein 90) and its telomerase activity is resistant to Hsp90 inhibitors, whereas the latter contains Hsp90 and its telomerase activity is sensitive to Hsp90 inhibitors. N-terminal of FLAG-hTERT in the former is exposed, as this complex was efficiently purified with anti-FLAG M2 affinity resin. We also identified two different telomerase complexes in HeLa cells, in addition to ectopically expressed hTERT. Most of endogenous hTERT and FLAG-hTERT was detected around 680 kDa. These two complexes in HeLa cells have the same properties as their respective reconstituted telomerases. The unstable property of the telomerase complex with Hsp90, especially in the presence of Hsp90 inhibitors, was due to proteasome-mediated degradation of hTERT, since proteasome inhibitors prevented hTERT degradation in vivo. To our knowledge, this is the first demonstration of two distinct active complexes of human telomerase ectopically expressed in insect and mammalian cells.

Heterogeneous nuclear ribonucleoprotein A2/B1 in association with hTERT is a potential biomarker for hepatocellular carcinoma

“…B,C). Hsp90 inhibitors reduce the amount of hTERT as well as telomerase activity. Despite the concentration of Hsp90 inhibitors, no telomere shortening of Hsp90 inhibitor‐treated cells was observed (data not shown).…”

Human Telomerase Domain Interactions Capture DNA for TEN Domain-Dependent Processive Elongation

“…Previous observations can be unified by proposing Hsp90 control of the TEN domain linker: drug inhibition or depletion of Hsp90 could affect the conformational freedom of the TEN domain to allow TRBD access to assembly with hTR, substrate access to the template, and release of enzyme-product interaction. Reconstituted telomerase RNPs may be most sensitive to Hsp90 inhibition if they assemble with a cis -docked rather than trans -docked TEN domain, accounting for the differential Hsp90 inhibitor dependence of distinct populations of reconstituted active human telomerase (Mizuno et al, 2007). …”

“…For example, human telomerase holoenzyme biogenesis must load a monomer of human telomerase RNA (hTR) with two complete sets of H/ACA RNP proteins before the RNP can recruit a monomer of TERT (Errington et al, 2008; Egan and Collins, 2010). Lacking the extensive cohort of chaperones and cofactors that provide assembly specificity in vivo , in vitro reconstitution of human TERT and hTR typically yields heterogeneous complexes with varying enzyme properties (Mizuno et al, 2007). …”

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

“…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. By adding a higher dose of GA, a complete inhibition of telomerase activity was observed.…”

“…Previous evidence indicates that telomere erosion plays a major role in cellular senescence, and the activation of telomerase extends the cellular lifespan via a stabilization of telomere lengths (Minamino and Komuro, 2003). Heat shock protein 90 (HSP90), a molecular chaperon, has been shown to be associated with TERT for sufficient telomerase activity, and the addition of HSP90 inhibitor geldanamycin (GA) prevents the formation of active telomerase via ubiquitination and proteasome-dependent degradation of TERT (Mizuno et al, 2007). These data suggest that HSP90 participates in telomerase function.…”