Leishmania major Telomerase TERT Protein Has a Nuclear/Mitochondrial Eclipsed Distribution That Is Affected by Oxidative Stress

Abstract

In its canonical role the reverse transcriptase telomerase recovers the telomeric repeats that are lost during DNA replication. Other locations and activities have been recently described for the telomerase protein subunit TERT in mammalian cells. In the present work, using biochemistry, molecular biology, and electron microscopy techniques, we found that in the human parasite Leishmania major, TERT (and telomerase activity) shared locations between the nuclear, mitochondrial, and cytoplasmic compartments. Also, some telomerase activity and TERT protein could be found in ϳ100-nm nanovesicles. In the mitochondrial compartment, TERT appears to be mainly associated with the kinetoplast DNA. When Leishmania cells were exposed to H 2 O 2 , TERT changed its relative abundance and activity between the nuclear and mitochondrial compartments, with the majority of activity residing in the mitochondrion. Finally, overexpression of TERT in Leishmania transfected cells not only increased the parasitic cell growth rate but also increased their resistance to oxidative stress. Mammalian telomeres are nucleoprotein complexes with double and single DNA strand (telomeric overhang) runs of TTAGGG repeats, a chromatin organization containing methylated H3 and H4 histones, and a heavily methylated DNA, which are features of a transcriptionally repressed zone. At the ends of linear chromosomes, since DNA polymerases do not initiate DNA synthesis de novo, the new DNA synthesized over the lagging strand lacks the primer for the completion of its synthesis, leaving a gap; how to seal this gap has been named the end replication problem (1). Most eukaryotes solve the problem using mechanisms in which the enzyme telomerase plays an important role (2). Telomerase has been described for most eukaryotic cells, and in fact, the first report of telomerase was made for the protozoan Tetrahymena (2). The telomerase core has two components: a reverse transcriptase (TERT) protein and an RNA subunit (TER) that provides the template for copying the hexameric repeats to the telomeric overhang (2). In addition, mammalian telomeres contain a six-protein complex called shelterin that regulates the telomerase activity and protects the chromosomal ends from nucleolytic degradation and chromosomal rearrangements (3).The TERT component has the typical motifs of reverse transcriptase enzymes (1, 2, A, B=, C, and E) and a telomerase-specific T-domain (4), whereas the TER subunit has been sequenced in a large number of organisms, and the length of the RNA varies from ϳ200 to 1,300 nucleotides (nt) (5).Growing evidence indicates that TERT plays multiple roles in addition to its telomeric function, including (i) acting as a transcriptional modulator of the ␤-catenin signaling pathway in mice; (ii) interacting with an RNA component of mitochondrial RNA processing endoribonuclease (RMRP), creating a new activity that synthetizes RNA in a RNA-dependent manner and generating a double-stranded RNA (dsRNA) that enters into the RNA interference (RNAi) cascade; (iii) regu...

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