ABSTRACT: Organization of the genome into compacted chromatin is a eukaryotic innovation facilitating increased sophistication in transcriptional regulation. In metazoa coiled-coil lamin proteins are major components of the chromatin organizer at the nuclear periphery and maintain nuclear integrity. While identifiable lamin homologues are restricted to metazoans, morphologically analogous structures maintaining nuclear organization in other eukaryotic lineages are known, but the molecular constituents remain undefined. Trypanosoma brucei NUP-1 is a large coiled-coil protein associated with fibrils at the inner face of the nuclear envelope. Using transcriptome analysis in combination with RNA interference and various imaging techniques, we demonstrate that NUP-1 forms a stable immobile cage around the nucleus, is required for viability and nuclear structural integrity, directs the positional organization of nuclear pore complexes, and serves to organize chromatin and specifically repress genes located at the nuclear periphery involved in immune evasion. Based on architectural similarity and functionality, we propose that NUP-1 is a novel, highly divergent lamin The effect of Nup-1 depletion on the transcriptome was examined in three independent experiments (A, B, & C). T. brucei cultures were either treated with RNAi (plus) or left untreated (minus) and RNA was extracted from each sample at the indicated time point (0h, 6h, 12h, 24h, or 48h). Two color microarrays were performed comparing treated and untreated samples at each time point. Dye swaps were performed and are indicated. Replicates of t=12h and t=24h for sample B were also included.
Project description:The enzyme N-myristoyltransferase (NMT) catalyses the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localisation of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labelling with an alkyne-tagged myristic acid analogue (“YnMyr”), enabling the capture of lipidated proteins in insect (PCF) and host (BSF) life stages of T. brucei. We further compare this with a longer chain palmitate analogue (“YnPal”) to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors (Cpds 1 and 2) and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites.
Project description:The nuclear lamina has multiple functions, including maintaining nuclear structural integrity and differential gene expression. Correct spatial and temporal lamina assembly is essential to meet these and other roles. Recently, it emerged that multiple lamina systems exist that are likely products of independent origins, while all these systems share remarkably analogous functions. Several lamina proteins are known in trypanosomes, two of which, NUP-1 and NUP-2, are essential, coiled-coil proteins with a molecular mass 450 and 250 kDa, respectively. Sequence analysis indicates distinct quaternary structures when compared to the ~60 kDa lamin proteins of multiple lineages, including metazoa. To uncover organisational principles of the trypanosome lamina we generated NUP-1 deletion mutants (N=N-terminal domain; C= C-terminal domain; NC: fusion of the N- and C-terminal domain with entire repeat region deletedd))designed to identify domains of NUP-1 responsible for oligomerisation. We find that both N- and C-termini act as interaction domains and disruption of these interactions impacts additional components of the lamina, the nuclear envelope and nucleoporin TbNup98. By contrast there is remarkably little impact on transcription, crucially including silencing of telomeric variant surface glycoprotein genes. These data indicate that both terminal domains of NUP-1 have roles in assembling the trypanosome lamina and suggest an architecture distinct to the lamin system is based on a ‘hub and spoke’ configuration.
Project description:Benzoxaboroles (BoBs) feature a boron-heterocyclic core and are an important recent innovation in the development of drugs against a range of pathogens and other pathologies. A broad spectrum of pharmacology is associated with chemically diverse BoB derivatives and includes multiple modes-of-action and targets. However, a consensus MoA for BoBs targeting evolutionarily diverse protozoan pathogens has emerged with the identification of CPSF3/CPSF73 in the CPSF complex in both apicomplexan and kinetoplastida parasites. We have detected a functional connection between protein sumoylation and the BoB boron-heterocyclic scaffold using comprehensive genetic screens in Trypanosoma brucei. Strikingly, as part of this sumoylation response, members of the CPSF complex are specifically and rapidly destabilised in a SUMO and proteosome-dependent manner. Here we deposit RNAseq data quantifying the effects of the aminomethyl-benzoxaborole AN3057 exposure on the transcriptome landscape in T. brucei. Specifically, T. brucei bloodstream-form cells in logarithmic growth phase were treated with 400 nm AN3057 (3 × EC50 determined after 24h) for 20 min (T20) and 60 min (T60), respectively. Nontreated control cells were prepared in parallel. All samples were in 2 biological replicates.
Project description:African trypanosomes are dixenous eukaryotic parasites that impose a significant human and veterinary disease burden on sub-Saharan Africa. Diversity between species and life-cycle stages is concomitant with distinct host and tissue tropisms within this group. Here, the spatial proteomes of two African trypanosome species, Trypanosoma brucei and Trypanosoma congolense, have been mapped, each in mammalian and insect life-stages represented by bloodstream form (BSF) and procyclic form (PCF) respectively. Using the hyperLOPIT (hyperplexed localisation of organelle proteins by isotope tagging) methodology, this work has provided four highly comprehensive spatial proteomes.
Project description:Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomics analyses of saliva from T. brucei-infected flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of GPIanchored surface proteins herein named Metacyclic Invariant Surface Proteins (MISP). The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are extended above the VSG coat. However, vaccination with recombinant MISP360 isoform did not protect mice against a T. brucei infectious tsetse bite. Lastly, both RNAi knock down and CRISPR-Cas9-driven knock out of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We speculate that MISP may be relevant during trypanosome inoculation or establishment in the vertebrate’s skin.
Project description:In a phenotypic screening approach of novel molecules composed of a synergistic combination of phthalimide, benzimidazole, and triazole scaffolds we discovered compounds with potent anti-leishmanial activity. The resulting early-lead compound PHT-39, which contains a trifluoromethyl substitution, demonstrated the highest efficacy in a Leishmania infantum intramacrophage assay, with an EC50 of 1.2+/- 3.2 μM.Cytotoxicity testing of PHT-39 in Hep-G2 cells indicated high selectivity of over 90-fold. To investigate the mechanism of action we carried out experiments in Trypanosoma brucei, which is also sensitive to PHT-39. Here we used a genome-wide RNAi library approach (PMID: 22278056; PMID: 21363968) to detect sensitivity determinants. This high-throughput phenotyping approach identified sensitivity determinants for PHT-39, which included a P-type ATPase that is crucial for the uptake of miltefosine and amphotericin, strongly indicating a shared route for cellular entry.
Project description:In trypanosomes two deubiquitilating enzymes (DUBs), orthologous to human USP7 and VDU1, control abundance of a cohort of surface proteins, including invariant surface glycoproteins (ISGs). Silencing TbUsp7 partially inhibits endocytosis and invariant surface glycoprotein turnover. S-phase kinase-associated protein 1 (Skp1) has crucial roles in cell cycle progression, transcriptional regulation, signal transduction and other processes in animals and fungi by virtue of being a component of cullin E3 ubiquitin ligases. Unexpectedly, trypanosomes possess multiple Skp1 paralogs, including a divergent paralog designated SkpZ. SkpZ is implicated in suramin-sensitivity and endocytosis and decreases in abundance following TbUsp7 knockdown. SkpZ physically interacts with TbUsp7 and TbTpr86. The latter is a tetratricopeptide-repeat protein also implicated in suramin sensitivity and located close to the flagellar pocket/endosomes, consistent with a role in endocytosis. Further, silencing SkpZ reduced abundance of TbUsp7 and TbTpr86 and many trans-membrane domain surface proteins. Our data indicate that TbTpr, TbUsp7 and SkpZ form the ‘TUS’ complex that regulates abundance of a significant cohort of trypanosome surface proteins.
Project description:Cellular proteins vary significantly in both abundance and turnover rates. These parameters depend upon their rates of synthesis and degradation and it is useful to have look-up data on protein turnover rates when, for example, designing genetic knock-down experiments or assessing the potential usefulness of covalent enzyme inhibitors. Little is known about the nature and regulation of protein turnover in Trypanosoma brucei, the etiological agent human and animal African trypanosomiasis.
Project description:The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organisation. In many eukaryotes the coiled coil Mlp/Tpr proteins of the NPC nuclear basket have specific roles in interactions with chromatin and defining specialised regions of active transcription, while Mlp2 associates with the mitotic spindle in a cell-cycle dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and low fidelity telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site, but apparently has minimal roles in the control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analog of Mlp/Tpr proteins, and together with the lamina analog NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina. Whole transcriptome comparison between parental and TbNup92? cells
Project description:As an additional strategy for investigating T. brucei transcriptome responsiveness, the mRNA of a highly important protein, the variant surface glycoprotein (VSG) the major surface protein in the bloodstream stage, was suppressed with RNAi. VSG RNAi results in arrest of cell cycle progression in the bloodstream stage. In addition, the mRNA of a highly important protein for endocytosis, the clathrin heavy chain (CLH), was suppressed with RNAi. CLH knockdown leads to a complete block to endocytosis. We hypothesized that if trypanosomes were able to sense alterations in trafficking and respond to these changes, then depletion of these ORFs by RNAi would be expected to elicit a response at the transcriptome level.<br> <br> part 1: 2 biological replicates of SMB cells transfected with the VSG- RNAi vector grown under normal conditions (non-induced), and 2 replicates of the same cells treated with 1 ug/ml tetracycline (induced) for 24hr, as well as dye swaps were used.<br> <br> part 2: 2 biological replicates of SMB cells transfected with the VSG- RNAi vector grown under normal conditions (non-induced), and 2 replicates of the same cells treated with 1 ug/ml tetracycline (induced) for 3 days, as well as dye swaps were used.<br> <br> part 3: 3 biological replicates of SMB cells transfected with the CLH- RNAi vector grown under normal conditions (non-induced), and 3 replicates of the same cells treated with 1 ug/ml tetracycline (induced), as well as dye swaps were used.