Proteomic analysis defines the interactome of telomerase in the protozoan parasite, Trypanosoma brucei

Abstract

Telomerase is a ribonucleoprotein enzyme responsible for maintaining the telomeric end of the chromosome. The telomerase enzyme requires two main components to function: the telomerase reverse transcriptase (TERT) and the telomerase RNA (TR), which provides the template for telomeric DNA synthesis. TR is a long non-coding RNA, which forms the basis of a large structural scaffold upon which many accessory proteins can bind and form the complete telomerase holoenzyme. These accessory protein interactions are required for telomerase activity and regulation inside cells. The interacting partners of TERT have been well studied in yeast, human, and Tetrahymena models, but not in parasitic protozoa, including clinically relevant human parasites. Here, using the protozoan parasite, Trypanosoma brucei (T. brucei) as a model, we have identified the interactome of T. brucei TERT (TbTERT) using a mass spectrometry-based approach. We identified previously known and unknown interacting factors of TbTERT, highlighting unique features of T. brucei telomerase biology. These unique interactions with TbTERT, suggest mechanistic differences in telomere maintenance between T. brucei and other eukaryotes.

Keywords: Trypanosoma brucei; cell growth; cell proliferation; interactome; parasite; ribonucleoprotein (RNP); telomerase; telomere.

FIGURE 1

Affinity-purification mass spectrometry of bloodstream form T. brucei telomerase reverse transcriptase. (A) Experimental workflow for proteomic analysis. BF T. brucei cells expressing a FLAG tagged version of TbTERT were grown and TbTERT complexes were purified using a custom anti-TbTERT C terminus antibody. An IgG isotype antibody was used as a control. Purified TbTERT complexes were then digested with trypsin. These peptides were then analyzed by LC-MS/MS. (B) Western blot confirming the presence of immunopurified TbTERT. IP samples were obtained and run on SDS-PAGE gels and immunoblotted with anti-FLAG antibody to detect TbTERT. (C) Western blot confirming the presence of TbTERT. IP samples were obtained and run on SDS-PAGE gels and immunoblotted with an anti-TbTERT C terminus antibody to detect TbTERT. (D) SDS-PAGE analysis of immunopurified TbTERT. A small aliquot was also resolved on SDS-PAGE and stained with Coomassie stain to qualitatively check TbTERT protein levels. (E) RT-qPCR detection of TbTR from Immunopurified TbTERT complexes. (F) Telomerase activity of the bead-bound telomerase enzyme was analyzed by telomerase primer extension assay.

FIGURE 2

(A) Volcano plot was performed with an x-axis representing the difference in logarithmic protein intensities between the TbTERT immunoprecipitation elution and the isotype matched IgG control (Elution and Control experimental groups). The y-axis is the negative log of the two-sided Student’s t-test. The volcano plot serves as a visual representation of the protein groups that are significantly enriched between the elution and control groups. These enriched groups contain the bait protein TbTERT and several candidates interacting with a p-value ≤ 0.05. (B) Protein-protein interaction network of relevant TbTERT hits identified by MS. Network was generated using the STRING database and visualized using Cytoscape. Colors of nodes represent the protein’s biological function. The thickness of the lines denotes the strength of the interaction (confidence PPI, threshold: 0.4, medium confidence).

FIGURE 3

GO analysis of T. brucei TERT interactome. STRING GO analysis (A) Enrichment by Biological process. The top 15 enriched GO terms are shown. (B) enrichment by Cellular component. The top 9 enriched GO terms are shown.

FIGURE 4

NOP58 interacts with the T. brucei telomerase complex. (A) Domain structure of human NOP58. Predicted secondary structure models for T. brucei and human NOP58 obtained from AlphaFold (Jumper et al., 2021) Predicted protein structures are shown in the same orientation. Dark blue represents a very high model confidence (pLDDT >90), light blue confident (90 > pLDDT >70), yellow low confidence (70 > pLDDT >50), orange very low confidence (pLDDT <50). Secondary structure model of T. brucei telomerase RNA. The C/D box binding motif is highlighted. [(B), top] Co-IP assay using WT T. brucei cell lysate. IP antibody: anti-TbTERT C terminus; Western blot antibodies: anti-FLAG and anti-NOP58. [(B), bottom] Co-IP using both WT and ∆C/D box mutant cells. IP antibody: anti-TbTERT C terminus; western blot antibodies: anti-FLAG and anti-NOP58.