The conserved hypothetical protein Tb427.10.13790 is required for cytokinesis in Trypanosoma brucei

Mudogo, Célestin Nzanzu; Werner, Salesia Franziska; Mogk, Stefan; Betzel, Christian; Duszenko, Michael

doi:10.1016/j.actatropica.2018.08.029

Cited by 2 publications

(2 citation statements)

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“…The characterization of novel proteins from human pathogenic trypanosomatids, such as T. cruzi, contributes to unveiling the unknown biological aspects of these parasites [70][71][72] and others. This, in turn, can lead to the discovery of new candidates for diagnoses, prognoses, or biomarkers for treatment cures, as well as a potential drug target [73].…”

Section: Discussionmentioning

confidence: 99%

Characterization of Novel Trypanosoma cruzi-Specific Antigen with Potential Use in the Diagnosis of Chagas Disease

Ossowski,

Gallardo,

Niborski

et al. 2024

IJMS

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Chagas disease is caused by the parasite Trypanosoma cruzi. In humans, it evolves into a chronic disease, eventually resulting in cardiac, digestive, and/or neurological disorders. In the present study, we characterized a novel T. cruzi antigen named Tc323 (TcCLB.504087.20), recognized by a single-chain monoclonal antibody (scFv 6B6) isolated from the B cells of patients with cardiomyopathy related to chronic Chagas disease. Tc323, a ~323 kDa protein, is an uncharacterized protein showing putative quinoprotein alcohol dehydrogenase-like domains. A computational molecular docking study revealed that the scFv 6B6 binds to an internal domain of Tc323. Immunofluorescence microscopy and Western Blot showed that Tc323 is expressed in the main developmental forms of T. cruzi, localized intracellularly and exhibiting a membrane-associated pattern. According to phylogenetic analysis, Tc323 is highly conserved throughout evolution in all the lineages of T. cruzi so far identified, but it is absent in Leishmania spp. and Trypanosoma brucei. Most interestingly, only plasma samples from patients infected with T. cruzi and those with mixed infection with Leishmania spp. reacted against Tc323. Collectively, our findings demonstrate that Tc323 is a promising candidate for the differential serodiagnosis of chronic Chagas disease in areas where T. cruzi and Leishmania spp. infections coexist.

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Section: Discussionmentioning

confidence: 99%

Characterization of Novel Trypanosoma cruzi-Specific Antigen with Potential Use in the Diagnosis of Chagas Disease

Ossowski,

Gallardo,

Niborski

et al. 2024

IJMS

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“…In these studies, when AEK1 function is abrogated, replication and segregation of the kinetoplast (the catenated mitochondrial DNA, kDNA) and nuclear DNA appear normal, but cytokinesis fails. The uncoupling of cell growth (enlargement), mitosis, and cytokinesis has been observed following genetic knockdown of several genes in the bloodstream form of T. brucei, for example Aurora kinase 1 (Jetton et al, 2009), variant surface glycoprotein (VSG) (Sheader et al, 2005), and the hypothetical protein Tb427.10.13790 (Mudogo et al, 2018). Chemical genetic analysis shows that 16 h after AEK1 inhibition approximately half of the parasites have aberrant DNA contents, so they are unlikely to give rise to functional daughter cells (Jensen et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

An essential Trypanosoma brucei protein kinase: a functional analysis of regulation and the identification of inhibitors

Parsons,

Dean

et al. 2023

Front. Parasitol.

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IntroductionThe protein serine/threonine kinase AEK1 is essential in the pathogenic stage of Trypanosoma brucei, the causative agent of African trypanosomiasis. AEK1 is a member of the AGC protein kinase family, although it is not closely related to a specific human AGC kinase. Our previous chemical genetic studies showed that targeted inhibition of AEK1 in parasites expressing analog-sensitive AEK1 blocked parasite growth and enhanced survival of infected mice.MethodsTo further validate AEK1 as a drug target, we used the chemical genetic system to determine the effect of a 24 hour loss of AEK1 activity on cell viability at the clonal level. A panel of 429 protein kinase inhibitors were screened against the wild-type protein for binding, using time-resolved fluorescence energy transfer (TR-FRET). The role of phosphorylation sites and motifs was probed by determining whether expression of proteins harboring mutations in these sequences could rescue AEK1 conditional knockout parasites. To determine the effect that mutations in the phosphosites have on the kinase activity of cellular AEK1 we compared the in vitro kinase activity of mutant and wild-type proteins immunoprecipitated from parasite lysates using the exogenous substrate MBP. Finally, the tagged AEK1 protein was localized by deconvolution microscopy.ResultsAfter a 24 hour exposure to an AEK1 inhibitory analog in the chemical genetic system, less than five percent of the remaining live cells can clonally expand, further validating AEK1 as a drug target. In the AEK1 inhibitor screening assay, we identified 17 hit compounds. Complementation studies showed that of the two known phosphorylation sites in the activation loop; mutation of one abolished function while mutation of the other had no discernable effect. Mutation of the other two AEK1 phosphosites gave intermediate phenotypes. Mutations in either the hydrophobic motif at the C-terminus of the protein or in the region of AEK1 predicted to bind the hydrophobic motif were also required for function. All parasites with defective AEK1 showed reduced proliferation and defects in cytokinesis, although the tested mutations differed in terms of the extent of cell death. Kinase activity of immunoprecipitated AEK1 phosphosite mutants largely paralleled the effects seen in complementation studies, although the mutation of the phosphosite adjacent to the hydrophobic motif had a greater impact on activity than predicted by the complementation studies. AEK1 was localized to cytoplasmic puncta distinct from glycosomes and acidocalcisomes.DiscussionThe rapid loss of viability of cells inhibited for AEK1 supports the idea that a short course of treatment that target AEK1 may be sufficient for treatment of people or animals infected with T. brucei. Key regulatory elements between AEK1 and its closest mammalian homolog appear to be largely conserved despite the vast evolutionary distance between mammals and T. brucei. The presence of AEK1 in cytoplasmic puncta raises the possibility that its localization may also play a role in functional activity.

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The conserved hypothetical protein Tb427.10.13790 is required for cytokinesis in Trypanosoma brucei

Cited by 2 publications

References 52 publications

Characterization of Novel Trypanosoma cruzi-Specific Antigen with Potential Use in the Diagnosis of Chagas Disease

Characterization of Novel Trypanosoma cruzi-Specific Antigen with Potential Use in the Diagnosis of Chagas Disease

An essential Trypanosoma brucei protein kinase: a functional analysis of regulation and the identification of inhibitors

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