Analysis of Leishmania GSK3 beta signalling pathways

According to the World Health Organisation (WHO), leishmaniasis belongs to the twenty-one neglected tropical diseases affecting over 1 million people each year in 98 countries worldwide. The mortality rate is estimated to be 70,000 annual deaths indicating the need of a treatment (Torres-Guerrero et al., 2017). Protein kinases are potential antileishmanial drug targets. GSK3β is involved in important cellular processes such as cell proliferation and apoptosis. LmxGSK3β has been proven essential for the survival of L. mexicana in the promastigote insect stage and amastigote mammalian stage of the parasite suggesting that this is a protein worth investigating further (Munro, 2013). To serve this scope, the present project aimed to confirm potential substrates of this protein. From a list with putative substrates of LmxGSK3β, which was already generated using BioID in our lab, LmxDynamin and LmxKIN13-1 were used for further analyses. Moreover, a peptide derived from LmxUBH5 was found to interact with LxmGSK3β in a kinase assay, thus this protein was included as a putative substrate in this investigation.;To address the hypothesis that LmxDynamin, LmxKIN13-1 and LmxUBH5 are bona fide LmxGSK3β substrates CRISPR/Cas9 technology and bioinformatics tools were utilised. Attempts to tag LmxDynamin with mNeonGreen (mNG) using CRISPR/Cas9 were unsuccessful. Likewise, a knockout of LmxUBH5 could not be obtained. It might be that LmxUBH5 is essential for the parasite and hence its deletion is not possible. CRISPR/Cas9 allowed C- and N- terminal mNG-fusion of LmxKIN13-1 and LmxUBH5, respectively. Diagnostic PCRs detected the correct integration of donor DNA fragments into the L. mexicana target gene loci. Immunoblot analysis confirmed expression of the full-length fusion proteins. LmxUBH5 was found in the cytoplasm of promastigotes in all stages of the cell cycle. Some of the transfected cells appeared with normal morphology, while some cells were affected by the tagging and showed an aberrant cell shape and their cytokinesis started from the posterior end of the cell, opposite from the flagellum. LmxKIN13-1 localised in the nucleus. It was only present during mitosis. Its homologue in Trypanosoma brucei, TbKIN13-1, showed similar results (Wickstead et al., 2010). A three-dimensional model of the LmxKIN13-1 motor domain was generated based on the structure of the Plasmodium falciparum kinesin pKinI (PDB 1ry6). The motor domain is at the centre of the protein classifying LmxKIN13-1 as an M-kinesin. It contains the conserved ATP-binding and microtubule interaction sites.;The ATP-binding site is comprised of the N1/P loop (GQTGSGKT), the N2/Switch I (NxxSSRSH), the N3/Switch II (DLAGxER) and N4 (RxRP). The microtubule interaction site consists of the α4-helix (KECIR), loop 2 (KVD), loop 8 (LRxLEDxKxxVxxGL) and loop 12 (QNKxHTPVSK), which are highly conserved among proteins of the kinesin-13 family in different Leishmania spp and in T. brucei. The central location of the motor domain is a general feature of kinesin13-1 family members. This study presents evidence that LmxKIN13-1 is involved in mitosis. Therefore, as a potential interaction partner of LmxGSK3β, LmxKIN13-1 activity might be regulated by this kinase allowing the development of a mitotic inhibitor. This could be then used to design a drug against leishmaniasis. Further analysis utilising Miniturbo-2C BioID and kinase assays with recombinantly expressed proteins were planned to investigate this aspect but could not be performed due to the COVID-19 pandemic.According to the World Health Organisation (WHO), leishmaniasis belongs to the twenty-one neglected tropical diseases affecting over 1 million people each year in 98 countries worldwide. The mortality rate is estimated to be 70,000 annual deaths indicating the need of a treatment (Torres-Guerrero et al., 2017). Protein kinases are potential antileishmanial drug targets. GSK3β is involved in important cellular processes such as cell proliferation and apoptosis. LmxGSK3β has been proven essential for the survival of L. mexicana in the promastigote insect stage and amastigote mammalian stage of the parasite suggesting that this is a protein worth investigating further (Munro, 2013). To serve this scope, the present project aimed to confirm potential substrates of this protein. From a list with putative substrates of LmxGSK3β, which was already generated using BioID in our lab, LmxDynamin and LmxKIN13-1 were used for further analyses. Moreover, a peptide derived from LmxUBH5 was found to interact with LxmGSK3β in a kinase assay, thus this protein was included as a putative substrate in this investigation.;To address the hypothesis that LmxDynamin, LmxKIN13-1 and LmxUBH5 are bona fide LmxGSK3β substrates CRISPR/Cas9 technology and bioinformatics tools were utilised. Attempts to tag LmxDynamin with mNeonGreen (mNG) using CRISPR/Cas9 were unsuccessful. Likewise, a knockout of LmxUBH5 could not be obtained. It might be that LmxUBH5 is essential for the parasite and hence its deletion is not possible. CRISPR/Cas9 allowed C- and N- terminal mNG-fusion of LmxKIN13-1 and LmxUBH5, respectively. Diagnostic PCRs detected the correct integration of donor DNA fragments into the L. mexicana target gene loci. Immunoblot analysis confirmed expression of the full-length fusion proteins. LmxUBH5 was found in the cytoplasm of promastigotes in all stages of the cell cycle. Some of the transfected cells appeared with normal morphology, while some cells were affected by the tagging and showed an aberrant cell shape and their cytokinesis started from the posterior end of the cell, opposite from the flagellum. LmxKIN13-1 localised in the nucleus. It was only present during mitosis. Its homologue in Trypanosoma brucei, TbKIN13-1, showed similar results (Wickstead et al., 2010). A three-dimensional model of the LmxKIN13-1 motor domain was generated based on the structure of the Plasmodium falciparum kinesin pKinI (PDB 1ry6). The motor domain is at the centre of the protein classifying LmxKIN13-1 as an M-kinesin. It contains the conserved ATP-binding and microtubule interaction sites.;The ATP-binding site is comprised of the N1/P loop (GQTGSGKT), the N2/Switch I (NxxSSRSH), the N3/Switch II (DLAGxER) and N4 (RxRP). The microtubule interaction site consists of the α4-helix (KECIR), loop 2 (KVD), loop 8 (LRxLEDxKxxVxxGL) and loop 12 (QNKxHTPVSK), which are highly conserved among proteins of the kinesin-13 family in different Leishmania spp and in T. brucei. The central location of the motor domain is a general feature of kinesin13-1 family members. This study presents evidence that LmxKIN13-1 is involved in mitosis. Therefore, as a potential interaction partner of LmxGSK3β, LmxKIN13-1 activity might be regulated by this kinase allowing the development of a mitotic inhibitor. This could be then used to design a drug against leishmaniasis. Further analysis utilising Miniturbo-2C BioID and kinase assays with recombinantly expressed proteins were planned to investigate this aspect but could not be performed due to the COVID-19 pandemic