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Functional characterisation and drug target validation of a mitotic kinesin-13 in Trypanosoma brucei.

ABSTRACT: Mitotic kinesins are essential for faithful chromosome segregation and cell proliferation. Therefore, in humans, kinesin motor proteins have been identified as anti-cancer drug targets and small molecule inhibitors are now tested in clinical studies. Phylogenetic analyses have assigned five of the approximately fifty kinesin motor proteins coded by Trypanosoma brucei genome to the Kinesin-13 family. Kinesins of this family have unusual biochemical properties because they do not transport cargo along microtubules but are able to depolymerise microtubules at their ends, therefore contributing to the regulation of microtubule length. In other eukaryotic genomes sequenced to date, only between one and three Kinesin-13s are present. We have used immunolocalisation, RNAi-mediated protein depletion, biochemical in vitro assays and a mouse model of infection to study the single mitotic Kinesin-13 in T. brucei. Subcellular localisation of all five T. brucei Kinesin-13s revealed distinct distributions, indicating that the expansion of this kinesin family in kinetoplastids is accompanied by functional diversification. Only a single kinesin (TbKif13-1) has a nuclear localisation. Using active, recombinant TbKif13-1 in in vitro assays we experimentally confirm the depolymerising properties of this kinesin. We analyse the biological function of TbKif13-1 by RNAi-mediated protein depletion and show its central role in regulating spindle assembly during mitosis. Absence of the protein leads to abnormally long and bent mitotic spindles, causing chromosome mis-segregation and cell death. RNAi-depletion in a mouse model of infection completely prevents infection with the parasite. Given its essential role in mitosis, proliferation and survival of the parasite and the availability of a simple in vitro activity assay, TbKif13-1 has been identified as an excellent potential drug target.

SUBMITTER: Chan KY

PROVIDER: S-EPMC2924347 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Functional characterisation and drug target validation of a mitotic kinesin-13 in Trypanosoma brucei.

Chan Kuan Yoow KY Matthews Keith R KR Ersfeld Klaus K

PLoS pathogens 20100819 8

Mitotic kinesins are essential for faithful chromosome segregation and cell proliferation. Therefore, in humans, kinesin motor proteins have been identified as anti-cancer drug targets and small molecule inhibitors are now tested in clinical studies. Phylogenetic analyses have assigned five of the approximately fifty kinesin motor proteins coded by Trypanosoma brucei genome to the Kinesin-13 family. Kinesins of this family have unusual biochemical properties because they do not transport cargo a ...[more]

PMID: 20808899

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Project description:BackgroundTrypanosomes can synthesize polyunsaturated fatty acids. Previously, we have shown that they possess stearoyl-CoA desaturase (SCD) and oleate desaturase (OD) to convert stearate (C18) into oleate (C18:1) and linoleate (C18:2), respectively. Here we examine if OD is essential to these parasites.MethodologyCultured procyclic (insect-stage) form (PCF) and bloodstream-form (BSF) Trypanosoma brucei cells were treated with 12- and 13-thiastearic acid (12-TS and 13-TS), inhibitors of OD, and the expression of the enzyme was knocked down by RNA interference. The phenotype of these cells was studied.Principal findingsGrowth of PCF T. brucei was totally inhibited by 100 µM of 12-TS and 13-TS, with EC(50) values of 40±2 and 30±2 µM, respectively. The BSF was more sensitive, with EC(50) values of 7±3 and 2±1 µM, respectively. This growth phenotype was due to the inhibitory effect of thiastearates on OD and, to a lesser extent, on SCD. The enzyme inhibition caused a drop in total unsaturated fatty-acid level of the cells, with a slight increase in oleate but a drastic decrease in linoleate level, most probably affecting membrane fluidity. After knocking down OD expression in PCF, the linoleate content was notably reduced, whereas that of oleate drastically increased, maintaining the total unsaturated fatty-acid level unchanged. Interestingly, the growth phenotype of the RNAi-induced cells was similar to that found for thiastearate-treated trypanosomes, with the former cells growing twofold slower than the latter ones, indicating that the linoleate content itself and not only fluidity could be essential for normal membrane functionality. A similar deleterious effect was found after RNAi in BSF, even with a mere 8% reduction of OD activity, indicating that its full activity is essential.Conclusions/significanceAs OD is essential for trypanosomes and is not present in mammalian cells, it is a promising target for chemotherapy of African trypanosomiasis.

Dataset Information

Functional characterisation and drug target validation of a mitotic kinesin-13 in Trypanosoma brucei.

Publications

Functional characterisation and drug target validation of a mitotic kinesin-13 in Trypanosoma brucei.

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