A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein

Leishmania spp. telomeres are composed of 5′-TTAGGG-3′ repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identi...

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Published in:Biochimica et biophysica acta. General subjects 2017-11, Vol.1861 (11), p.2583-2597
Main Authors: Morea, Edna G.O., Viviescas, Maria Alejandra, Fernandes, Carlos A.H., Matioli, Fabio F., Lira, Cristina B.B., Fernandez, Maribel F., Moraes, Barbara S., da Silva, Marcelo S., Storti, Camila B., Fontes, Marcos R.M., Cano, Maria Isabel N.
Format: Article
Language:English
Subjects:
Amino Acid Sequence - genetics
Calcium
Calmodulin
Calmodulin - chemistry
Calmodulin - genetics
DNA, Single-Stranded - chemistry
DNA, Single-Stranded - genetics
Humans
Leishmania - genetics
Leishmania - pathogenicity
Leishmania amazonensis
Leishmaniasis - genetics
Leishmaniasis - parasitology
Protein Binding
Telomerase
Telomere
Telomere end-binding protein
Telomere-Binding Proteins - chemistry
Telomere-Binding Proteins - genetics
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Summary:Leishmania spp. telomeres are composed of 5′-TTAGGG-3′ repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3′G-overhang, and DNA binding is calcium dependent. We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. LCalA is the first reported calmodulin that binds in vivo telomeric DNA. [Display omitted] •The protein component of LaGT1 was affinity-purified using the G-rich telomeric DNA.•The protein component of LaGT1 is a putative calmodulin, named LCalA.•LCalA is nuclear, partially co-localizes and binds in vivo the G-rich telomeric strand.•rLCalA binds calcium and the G-rich strand inducing protein structural changes.•rLCalA binds DNA with relative affinity and binding is calcium dependent.
ISSN:0304-4165
1872-8006
DOI:10.1016/j.bbagen.2017.08.011