Publications by authors named "Mark C Field"

180 Publications

A novel membrane complex is required for docking and regulated exocytosis of lysosome-related organelles in Tetrahymena thermophila.

PLoS Genet 2022 May 19;18(5):e1010194. Epub 2022 May 19.

Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois, United State of America.

In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.
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http://dx.doi.org/10.1371/journal.pgen.1010194DOI Listing
May 2022

CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes.

Mol Biochem Parasitol 2022 Apr 1;249:111476. Epub 2022 Apr 1.

The Wellcome Trust Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Electronic address:

Proteins of interest are frequently expressed with a fusion-tag to facilitate experimental analysis. In trypanosomatids, which are typically diploid, a tag-encoding DNA fragment is typically fused to one native allele. However, since recombinant cells represent ≪0.1% of the population following transfection, these DNA fragments also incorporate a marker cassette for positive selection. Consequently, native mRNA untranslated regions (UTRs) are replaced, potentially perturbing gene expression; in trypanosomatids, UTRs often impact gene expression in the context of widespread and constitutive polycistronic transcription. We sought to develop a tagging strategy that preserves native UTRs in bloodstream-form African trypanosomes, and here we describe a CRISPR/Cas9-based knock-in approach to drive precise and marker-free tagging of essential genes. Using simple tag-encoding amplicons, we tagged four proteins: a histone acetyltransferase, HAT2; a histone deacetylase, HDAC3; a cleavage and polyadenylation specificity factor, CPSF3; and a variant surface glycoprotein exclusion factor, VEX2. The approach maintained the native UTRs and yielded clonal strains expressing functional recombinant proteins, typically with both alleles tagged. We demonstrate utility for both immunofluorescence-based localisation and for enriching protein complexes; HAT2 or HDAC3 complexes in this case. This precision tagging approach facilitates the assembly of strains expressing essential recombinant genes with their native UTRs preserved.
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http://dx.doi.org/10.1016/j.molbiopara.2022.111476DOI Listing
April 2022

Proteomics Uncovers Novel Components of an Interactive Protein Network Supporting RNA Export in Trypanosomes.

Mol Cell Proteomics 2022 Mar 26;21(3):100208. Epub 2022 Jan 26.

Instituto Carlos Chagas, FIOCRUZ, Curitiba, Paraná, Brazil. Electronic address:

In trypanosomatids, transcription is polycistronic and all mRNAs are processed by trans-splicing, with export mediated by noncanonical mechanisms. Although mRNA export is central to gene regulation and expression, few orthologs of proteins involved in mRNA export in higher eukaryotes are detectable in trypanosome genomes, necessitating direct identification of protein components. We previously described conserved mRNA export pathway components in Trypanosoma cruzi, including orthologs of Sub2, a component of the TREX complex, and eIF4AIII (previously Hel45), a core component of the exon junction complex (EJC). Here, we searched for protein interactors of both proteins using cryomilling and mass spectrometry. Significant overlap between TcSub2 and TceIF4AIII-interacting protein cohorts suggests that both proteins associate with similar machinery. We identified several interactions with conserved core components of the EJC and multiple additional complexes, together with proteins specific to trypanosomatids. Additional immunoisolations of kinetoplastid-specific proteins both validated and extended the superinteractome, which is capable of supporting RNA processing from splicing through to nuclear export and cytoplasmic events. We also suggest that only proteomics is powerful enough to uncover the high connectivity between multiple aspects of mRNA metabolism and to uncover kinetoplastid-specific components that create a unique amalgam to support trypanosome mRNA maturation.
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http://dx.doi.org/10.1016/j.mcpro.2022.100208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8938319PMC
March 2022

Automated Phylogenetic Analysis Using Best Reciprocal BLAST.

Methods Mol Biol 2021 ;2369:41-63

Wellcome Centre for Anti-infectives Research, School of Life Sciences, University of Dundee, Dundee, UK.

Reconstruction of the evolutionary history of specific protein-coding genes is an essential component of the biological sciences toolkit and relies on identification of orthologs (a gene in different organisms related by vertical descent from a common ancestor and usually presumed to have the same or similar function) and paralogs (a gene related to another in the same organism by descent from a single ancestral gene which may, or may not, retain the same/similar function) across a range of taxa. While obviously essential for the reconstruction of evolutionary histories, ortholog identification is of importance for protein expression, modeling for drug discovery programs, identification of critical residues and other studies. Here we describe an automated system for searching for orthologs and paralogs in eukaryotic organisms. Unlike manual methods the system is fast, requiring minimal user input while still being highly configurable.
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http://dx.doi.org/10.1007/978-1-0716-1681-9_4DOI Listing
January 2022

The distinctive flagellar proteome of Euglena gracilis illuminates the complexities of protistan flagella adaptation.

New Phytol 2021 11 16;232(3):1323-1336. Epub 2021 Aug 16.

Institute of Parasitology, Biology Centre, Czech Academy of Sciences, České Budějovice (Budweis), 370 05, Czech Republic.

The eukaryotic flagellum/cilium is a prominent organelle with conserved structure and diverse functions. Euglena gracilis, a photosynthetic and highly adaptable protist, employs its flagella for both locomotion and environmental sensing. Using proteomics of isolated E. gracilis flagella we identify nearly 1700 protein groups, which challenges previous estimates of the protein complexity of motile eukaryotic flagella. We not only identified several unexpected similarities shared with mammalian flagella, including an entire glycolytic pathway and proteasome, but also document a vast array of flagella-based signal transduction components that coordinate gravitaxis and phototactic motility. By contrast, the pellicle was found to consist of > 900 protein groups, containing additional structural and signalling components. Our data identify significant adaptations within the E. gracilis flagellum, many of which are clearly linked to the highly flexible lifestyle.
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http://dx.doi.org/10.1111/nph.17638DOI Listing
November 2021

Evolution and diversification of the nuclear pore complex.

Biochem Soc Trans 2021 08;49(4):1601-1619

School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.

The nuclear pore complex (NPC) is responsible for transport between the cytoplasm and nucleoplasm and one of the more intricate structures of eukaryotic cells. Typically composed of over 300 polypeptides, the NPC shares evolutionary origins with endo-membrane and intraflagellar transport system complexes. The modern NPC was fully established by the time of the last eukaryotic common ancestor and, hence, prior to eukaryote diversification. Despite the complexity, the NPC structure is surprisingly flexible with considerable variation between lineages. Here, we review diversification of the NPC in major taxa in view of recent advances in genomic and structural characterisation of plant, protist and nucleomorph NPCs and discuss the implications for NPC evolution. Furthermore, we highlight these changes in the context of mRNA export and consider how this process may have influenced NPC diversity. We reveal the NPC as a platform for continual evolution and adaptation.
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http://dx.doi.org/10.1042/BST20200570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8421043PMC
August 2021

A hub-and-spoke nuclear lamina architecture in trypanosomes.

J Cell Sci 2021 06 21;134(12). Epub 2021 Jun 21.

School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

The nuclear lamina supports many functions, including maintaining nuclear structure and gene expression control, and correct spatio-temporal assembly is vital to meet these activities. Recently, multiple lamina systems have been described that, despite independent evolutionary origins, share analogous functions. In trypanosomatids the two known lamina proteins, NUP-1 and NUP-2, have molecular masses of 450 and 170 kDa, respectively, which demands a distinct architecture from the ∼60 kDa lamin-based system of metazoa and other lineages. To uncover organizational principles for the trypanosome lamina we generated NUP-1 deletion mutants to identify domains and their arrangements responsible for oligomerization. We found that both the N- and C-termini act as interaction hubs, and that perturbation of these interactions impacts additional components of the lamina and nuclear envelope. Furthermore, the assembly of NUP-1 terminal domains suggests intrinsic organizational capacity. Remarkably, there is little impact on silencing of telomeric variant surface glycoprotein genes. We suggest that both terminal domains of NUP-1 have roles in assembling the trypanosome lamina and propose a novel architecture based on a hub-and-spoke configuration.
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http://dx.doi.org/10.1242/jcs.251264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8255026PMC
June 2021

Kinetoplastid cell biology and genetics, from the 2020 British Society for Parasitology Trypanosomiasis and Leishmaniasis symposium, Granada, Spain.

Parasitology 2021 09 14;148(10):1119-1124. Epub 2021 Jun 14.

CNRS, Microbiology Fundamental and Pathogenicity, UMR 5234, F-33000Bordeaux, France.

The British Society for Parasitology (BSP) holds a biannual symposium devoted to the kinetoplastids, and seeks to cover the full gamut of research into these important organisms, and alternates with the Woods Hole Kinetoplastid Molecular Cell Biology meeting that serves a similar community. While normally embedded within the main BSP Spring meeting, on several occasions the symposium has enjoyed the opportunity of being hosted on mainland Europe. In 2020, the BSP was fortunate to spend some time in Granada in Spain, where a superb meeting with excellent science in a spectacular setting was overshadowed by news of an emerging novel coronavirus. In this editorial, we hope to have captured some of that excellent science and to highlight aspects of the many great papers and reviews in this special issue, as well as provide a few images from the meeting, which we hope for this who attended will bring back some fond memories.
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http://dx.doi.org/10.1017/S0031182021000998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8311962PMC
September 2021

Expression in Escherichia coli, purification and kinetic characterization of LAPLm, a Leishmania major M17-aminopeptidase.

Protein Expr Purif 2021 07 25;183:105877. Epub 2021 Mar 25.

Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, Calle 25 #455 Entre I y J, Vedado, 10400, Havana, Cuba. Electronic address:

The Leishmania major leucyl-aminopeptidase (LAPLm), a member of the M17 family of proteases, is a potential drug target for treatment of leishmaniasis. To better characterize enzyme properties, recombinant LAPLm (rLAPLm) was expressed in Escherichia coli. A LAPLm gene was designed, codon-optimized for expression in E. coli, synthesized and cloned into the pET-15b vector. Production of rLAPLm in E. coli Lemo21(DE3), induced for 4 h at 37 °C with 400 μM IPTG and 250 μM l-rhamnose, yielded insoluble enzyme with a low proportion of soluble and active protein, only detected by an anti-His antibody-based western-blot. rLAPLm was purified in a single step by immobilized metal ion affinity chromatography. rLAPLm was obtained with a purity of ~10% and a volumetric yield of 2.5 mg per liter, sufficient for further characterization. The aminopeptidase exhibits optimal activity at pH 7.0 and a substrate preference for Leu-p-nitroanilide (appK = 30 μM, appk = 14.7 s). Optimal temperature is 50 °C, and the enzyme is insensitive to 4 mM Co, Mg, Ca and Ba. However, rLAPLm was activated by Zn, Mn and Cd but is insensitive towards the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, being inhibited by EDTA and bestatin. Bestatin is a potent, non-competitive inhibitor of the enzyme with a K value of 994 nM. We suggest that rLAPLm is a suitable target for inhibitor identification.
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http://dx.doi.org/10.1016/j.pep.2021.105877DOI Listing
July 2021

Evolution, function and roles in drug sensitivity of trypanosome aquaglyceroporins.

Parasitology 2021 09 19;148(10):1137-1142. Epub 2021 Feb 19.

School of Life Sciences, University of Dundee, DundeeDD1 5EH, UK.

Aquaglyceroporins (AQPs) are membrane proteins that function in osmoregulation and the uptake of low molecular weight solutes, in particular glycerol and urea. The AQP family is highly conserved, with two major subfamilies having arisen very early in prokaryote evolution and retained by eukaryotes. A complex evolutionary history indicates multiple lineage-specific expansions, losses and not uncommonly a complete loss. Consequently, the AQP family is highly evolvable and has been associated with significant events in life on Earth. In the African trypanosomes, a role for the AQP2 paralogue, in sensitivity to two chemotherapeutic agents, pentamidine and melarsoprol, is well established, albeit with the mechanisms for cell entry and resistance unclear until very recently. Here, we discuss AQP evolution, structure and mechanisms by which AQPs impact drug sensitivity, suggesting that AQP2 stability is highly sensitive to mutation while serving as the major uptake pathway for pentamidine.
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http://dx.doi.org/10.1017/S0031182021000354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8311954PMC
September 2021

Evolution and diversification of the nuclear envelope.

Nucleus 2021 12;12(1):21-41

Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee , Dundee, UK.

Eukaryotic cells arose ~1.5 billion years ago, with the endomembrane system a central feature, facilitating evolution of intracellular compartments. Endomembranes include the nuclear envelope (NE) dividing the cytoplasm and nucleoplasm. The NE possesses universal features: a double lipid bilayer membrane, nuclear pore complexes (NPCs), and continuity with the endoplasmic reticulum, indicating common evolutionary origin. However, levels of specialization between lineages remains unclear, despite distinct mechanisms underpinning various nuclear activities. Several distinct modes of molecular evolution facilitate organellar diversification and  to understand which apply to the NE, we exploited proteomic datasets of purified nuclear envelopes from model systems for comparative analysis. We find enrichment of core nuclear functions amongst the widely conserved proteins to be less numerous than lineage-specific cohorts, but enriched in core nuclear functions. This, together with consideration of additional evidence, suggests that, despite a common origin, the NE has evolved as a highly diverse organelle with significant lineage-specific functionality.
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http://dx.doi.org/10.1080/19491034.2021.1874135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889174PMC
December 2021

Evolving Differentiation in African Trypanosomes.

Trends Parasitol 2021 04 11;37(4):296-303. Epub 2020 Dec 11.

School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK; Institute of Parasitology, Biology Centre, Czech Academy of Sciences, 37005 Ceske Budejovice, Czech Republic. Electronic address:

Differentiation is a central aspect of the parasite life cycle and encompasses adaptation to both host and environment. If we accept that evolution cannot anticipate an organism's needs as it enters a new environment, how do parasite differentiation pathways arise? The transition between vertebrate and insect stage African trypanosomes is probably one of the better studied and involves a cell-cycle arrested or 'stumpy' form that activates metabolic pathways advantageous to the parasite in the insect host. However, a range of stimuli and stress conditions can trigger similar changes, leading to formation of stumpy-like cellular states. We propose that the origin and optimisation of this differentiation program represents repurposing of a generic stress response to gain considerable gain-of-fitness associated with parasite transmission.
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http://dx.doi.org/10.1016/j.pt.2020.11.003DOI Listing
April 2021

Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs.

PLoS Pathog 2020 11 3;16(11):e1008932. Epub 2020 Nov 3.

Wellcome Centre for Integrative Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom.

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.
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http://dx.doi.org/10.1371/journal.ppat.1008932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710103PMC
November 2020

Reductionist Pathways for Parasitism in Euglenozoans? Expanded Datasets Provide New Insights.

Trends Parasitol 2021 02 27;37(2):100-116. Epub 2020 Oct 27.

Biology Centre, Institute of Parasitology, Czech Academy of Sciences, České Budějovice (Budweis), Czech Republic; Faculty of Sciences, University of South Bohemia, České Budějovice (Budweis), Czech Republic. Electronic address:

The unicellular trypanosomatids belong to the phylum Euglenozoa and all known species are obligate parasites. Distinct lineages infect plants, invertebrates, and vertebrates, including humans. Genome data for marine diplonemids, together with freshwater euglenids and free-living kinetoplastids, the closest known nonparasitic relatives to trypanosomatids, recently became available. Robust phylogenetic reconstructions across Euglenozoa are now possible and place the results of parasite-focused studies into an evolutionary context. Here we discuss recent advances in identifying the factors shaping the evolution of Euglenozoa, focusing on ancestral features generally considered parasite-specific. Remarkably, most of these predate the transition(s) to parasitism, suggesting that the presence of certain preconditions makes a significant lifestyle change more likely.
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http://dx.doi.org/10.1016/j.pt.2020.10.001DOI Listing
February 2021

Instability of aquaglyceroporin (AQP) 2 contributes to drug resistance in Trypanosoma brucei.

PLoS Negl Trop Dis 2020 07 9;14(7):e0008458. Epub 2020 Jul 9.

School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.

Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. Sensitivity of African trypanosomes to pentamidine and melarsoprol is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation across the plasma membrane or via binding to TbAQP2, with subsequent endocytosis and presumably transport across the endosomal/lysosomal membrane, but as trafficking and regulation of TbAQPs is uncharacterised this remains unresolved. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and is transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmic-oriented lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation, as observed in pentamidine-resistant parasites, also leads to impaired oligomerisation, mislocalisation and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and that instability contributes towards the emergence of drug resistance.
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http://dx.doi.org/10.1371/journal.pntd.0008458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413563PMC
July 2020

EIF2α phosphorylation is regulated in intracellular amastigotes for the generation of infective Trypanosoma cruzi trypomastigote forms.

Cell Microbiol 2020 11 28;22(11):e13243. Epub 2020 Jul 28.

Departmento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.

Trypanosomatids regulate gene expression mainly at the post-transcriptional level through processing, exporting and stabilising mRNA and control of translation. In most eukaryotes, protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2) at serine 51. Phosphorylation halts overall translation by decreasing availability of initiator tRNA to form translating ribosomes. In trypanosomatids, the N-terminus of eIF2α is extended with threonine 169 the homologous phosphorylated residue. Here, we evaluated whether eIF2α phosphorylation varies during the Trypanosoma cruzi life cycle, the etiological agent of Chagas' disease. Total levels of eIF2α are diminished in infective and non-replicative trypomastigotes compared with proliferative forms from the intestine of the insect vector or amastigotes from mammalian cells, consistent with decreased protein synthesis reported in infective forms. eIF2α phosphorylation increases in proliferative intracellular forms prior to differentiation into trypomastigotes. Parasites overexpressing eIF2α or with an endogenous CRISPR/Cas9-generated eIF2α mutation were created and analysis revealed alterations to the proteome, largely unrelated to the presence of μORF in epimastigotes. eIF2α mutant parasites produced fewer trypomastigotes with lower infectivity than wild type, with increased levels of sialylated mucins and oligomannose glycoproteins, and decreased galactofuranose epitopes and the surface protease GP63 on the cell surface. We conclude that eIF2α expression and phosphorylation levels affect proteins relevant for intracellular progression of T. cruzi.
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http://dx.doi.org/10.1111/cmi.13243DOI Listing
November 2020

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Using RapidFire Mass Spectrometry.

SLAS Discov 2020 10 13;25(9):1064-1071. Epub 2020 May 13.

Centre for Protein Studies, Faculty of Biology, University of Havana, La Habana, Cuba.

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC = 0.35 μM) and arphamenine A (IC = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.
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http://dx.doi.org/10.1177/2472555220923367DOI Listing
October 2020

Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes.

J Biol Chem 2020 06 30;295(24):8331-8347. Epub 2020 Apr 30.

School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite , occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism ( polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.
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http://dx.doi.org/10.1074/jbc.RA120.012355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294092PMC
June 2020

Sorting the Muck from the Brass: Analysis of Protein Complexes and Cell Lysates.

Methods Mol Biol 2020 ;2116:645-653

School of Life Sciences, University of Dundee, Dundee, UK.

Reliable determination of protein complex composition or changes to protein levels in whole cells is challenging. Despite the multitude of methods now available for labeling, analysis, and the statistical processing of data, this large variety is of itself an issue: Which approach is most appropriate, where do you set cutoffs, and what is the most cost-effective strategy? One size does not fit all for such work, but some guidelines can help in terms of reducing cost, improving data quality, and ultimately advancing investigations. Here we describe two protocols and algorithms for facile sample preparation for mass spectrometric analysis, robust data processing, and considerations of how to interpret large proteomic datasets in a productive and robust manner.
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http://dx.doi.org/10.1007/978-1-0716-0294-2_38DOI Listing
February 2021

A Uniquely Complex Mitochondrial Proteome from Euglena gracilis.

Mol Biol Evol 2020 08;37(8):2173-2191

Biology Centre, Institute of Parasitology, Czech Academy of Sciences, České Budějovice, Budweis, Czech Republic.

Euglena gracilis is a metabolically flexible, photosynthetic, and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free liquid chromatography tandem mass spectrometry, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing, and processing in the sister clades diplonemids and kinetoplastids is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins, and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.
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http://dx.doi.org/10.1093/molbev/msaa061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403612PMC
August 2020

Expression of a specific variant surface glycoprotein has a major impact on suramin sensitivity and endocytosis in .

FASEB Bioadv 2019 Oct 30;1(10):595-608. Epub 2019 Sep 30.

Swiss Tropical and Public Health Institute Basel Switzerland.

Suramin was introduced into the clinic a century ago and is still used to treat the first stage of acute human sleeping sickness. Due to its size and sixfold negative charge, uptake is mediated through endocytosis and the suramin receptor in trypanosomes is thought to be the invariant surface glycoprotein 75 (ISG75). Nevertheless, we recently identified a variant surface glycoprotein (VSG) that confers strong in vitro resistance to suramin in a line. In this study, we introduced into the active bloodstream expression site of a line. This caused suramin resistance and cross resistance to trypan blue. We quantified the endocytosis of different substrates by flow cytometry and showed that the expression of VSG strongly impairs the uptake of low-density lipoprotein (LDL) and transferrin, both imported by receptor-mediated endocytosis. However, bulk endocytosis and endocytosis of the trypanolytic factor were not affected, and the -expressors did not exhibit a growth phenotype in the absence of suramin. Knockdown of ISG75 was synergistic with expression, indicating that these two proteins are mediating distinct suramin resistance pathways. In conclusion, VSG causes suramin resistance in bloodstream forms by decreasing specific, receptor-mediated endocytosis pathways.
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http://dx.doi.org/10.1096/fba.2019-00033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996322PMC
October 2019

The Artemisinin Susceptibility-Associated AP-2 Adaptin μ Subunit is Clathrin Independent and Essential for Schizont Maturation.

mBio 2020 02 25;11(1). Epub 2020 Feb 25.

Department of Infection Biology, Faculty of Infectious Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of to artemisinin, associated with mutations in Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in , we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent. We examine in detail the AP-2 adaptin complex from the malaria parasite In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.
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http://dx.doi.org/10.1128/mBio.02918-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042695PMC
February 2020

Diversification of CORVET tethers facilitates transport complexity in .

J Cell Sci 2020 02 12;133(3). Epub 2020 Feb 12.

Department of Molecular Genetics and Cell Biology, 920 E 58th Street, The University of Chicago, Chicago, IL, 60637, USA

In endolysosomal networks, two hetero-hexameric tethers called HOPS and CORVET are found widely throughout eukaryotes. The unicellular ciliate possesses elaborate endolysosomal structures, but curiously both it and related protozoa lack the HOPS tether and several other trafficking proteins, while retaining the related CORVET complex. Here, we show that encodes multiple paralogs of most CORVET subunits, which assemble into six distinct complexes. Each complex has a unique subunit composition and, significantly, shows unique localization, indicating participation in distinct pathways. One pair of complexes differ by a single subunit (Vps8), but have late endosomal versus recycling endosome locations. While Vps8 subunits are thus prime determinants for targeting and functional specificity, determinants exist on all subunits except Vps11. This unprecedented expansion and diversification of CORVET provides a potent example of tether flexibility, and illustrates how 'backfilling' following secondary losses of trafficking genes can provide a mechanism for evolution of new pathways.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.238659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033735PMC
February 2020

Evolution of late steps in exocytosis: conservation and specialization of the exocyst complex.

Wellcome Open Res 2019 29;4:112. Epub 2019 Nov 29.

School of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK.

The eukaryotic endomembrane system most likely arose paralogous expansions of genes encoding proteins that specify organelle identity, coat complexes and govern fusion specificity. While the majority of these gene families were established by the time of the last eukaryotic common ancestor (LECA), subsequent evolutionary events has moulded these systems, likely reflecting adaptations retained for increased fitness. As well as sequence evolution, these adaptations include loss of otherwise canonical components, the emergence of lineage-specific proteins and paralog expansion. The exocyst complex is involved in late exocytosis and additional trafficking pathways and a member of the complexes associated with tethering containing helical rods (CATCHR) tethering complex family. CATCHR includes the conserved oligomeric Golgi (COG) complex, homotypic fusion and vacuole protein sorting (HOPS)/class C core vacuole/endosome tethering (CORVET) complexes and several others. The exocyst is integrated into a complex GTPase signalling network in animals, fungi and other lineages. Prompted by discovery of Exo99, a non-canonical subunit in the excavate protist and availability of significantly increased genome sequence data, we re-examined evolution of the exocyst. We examined the evolution of exocyst components by comparative genomics, phylogenetics and structure prediction. The exocyst composition is highly conserved, but with substantial losses of subunits in the Apicomplexa and expansions in Streptophyta plants, Metazoa and land plants, where for the latter, massive paralog expansion of Exo70 represents an extreme and unique example. Significantly, few taxa retain a partial complex, suggesting that, in general, all subunits are probably required for functionality. Further, the ninth exocyst subunit, Exo99, is specific to the Euglenozoa with a distinct architecture compared to the other subunits and which possibly represents a coat system. These data reveal a remarkable degree of evolutionary flexibility within the exocyst complex, suggesting significant diversity in exocytosis mechanisms.
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http://dx.doi.org/10.12688/wellcomeopenres.15142.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784791PMC
November 2019

SUMOylated SNF2PH promotes variant surface glycoprotein expression in bloodstream trypanosomes.

EMBO Rep 2019 12 6;20(12):e48029. Epub 2019 Nov 6.

Instituto de Parasitología y Biomedicina "López-Neyra", CSIC (IPBLN-CSIC), Granada, Spain.

SUMOylation is a post-translational modification that positively regulates monoallelic expression of the trypanosome variant surface glycoprotein (VSG). The presence of a highly SUMOylated focus associated with the nuclear body, where the VSG gene is transcribed, further suggests an important role of SUMOylation in regulating VSG expression. Here, we show that SNF2PH, a SUMOylated plant homeodomain (PH)-transcription factor, is upregulated in the bloodstream form of the parasite and enriched at the active VSG telomere. SUMOylation promotes the recruitment of SNF2PH to the VSG promoter, where it is required to maintain RNA polymerase I and thus to regulate VSG transcript levels. Further, ectopic overexpression of SNF2PH in insect forms, but not of a mutant lacking the PH domain, induces the expression of bloodstream stage-specific surface proteins. These data suggest that SNF2PH SUMOylation positively regulates VSG monoallelic transcription, while the PH domain is required for the expression of bloodstream-specific surface proteins. Thus, SNF2PH functions as a positive activator, linking expression of infective form surface proteins and VSG regulation, thereby acting as a major regulator of pathogenicity.
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http://dx.doi.org/10.15252/embr.201948029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893287PMC
December 2019

Metabolic quirks and the colourful history of the Euglena gracilis secondary plastid.

New Phytol 2020 02 4;225(4):1578-1592. Epub 2019 Nov 4.

Faculty of Science, Charles University, BIOCEV, Vestec, 252 50, Czechia.

Euglena spp. are phototrophic flagellates with considerable ecological presence and impact. Euglena gracilis harbours secondary green plastids, but an incompletely characterised proteome precludes accurate understanding of both plastid function and evolutionary history. Using subcellular fractionation, an improved sequence database and MS we determined the composition, evolutionary relationships and hence predicted functions of the E. gracilis plastid proteome. We confidently identified 1345 distinct plastid protein groups and found that at least 100 proteins represent horizontal acquisitions from organisms other than green algae or prokaryotes. Metabolic reconstruction confirmed previously studied/predicted enzymes/pathways and provided evidence for multiple unusual features, including uncoupling of carotenoid and phytol metabolism, a limited role in amino acid metabolism, and dual sets of the SUF pathway for FeS cluster assembly, one of which was acquired by lateral gene transfer from Chlamydiae. Plastid paralogues of trafficking-associated proteins potentially mediating fusion of transport vesicles with the outermost plastid membrane were identified, together with derlin-related proteins, potential translocases across the middle membrane, and an extremely simplified TIC complex. The Euglena plastid, as the product of many genomes, combines novel and conserved features of metabolism and transport.
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http://dx.doi.org/10.1111/nph.16237DOI Listing
February 2020

Monoallelic expression and epigenetic inheritance sustained by a Trypanosoma brucei variant surface glycoprotein exclusion complex.

Nat Commun 2019 07 9;10(1):3023. Epub 2019 Jul 9.

The Wellcome Trust Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK.

The largest gene families in eukaryotes are subject to allelic exclusion, but mechanisms underpinning single allele selection and inheritance remain unclear. Here, we describe a protein complex sustaining variant surface glycoprotein (VSG) allelic exclusion and antigenic variation in Trypanosoma brucei parasites. The VSG-exclusion-1 (VEX1) protein binds both telomeric VSG-associated chromatin and VEX2, an ortholog of nonsense-mediated-decay helicase, UPF1. VEX1 and VEX2 assemble in an RNA polymerase-I transcription-dependent manner and sustain the active, subtelomeric VSG-associated transcription compartment. VSG transcripts and VSG coats become highly heterogeneous when VEX proteins are depleted. Further, the DNA replication-associated chromatin assembly factor, CAF-1, binds to and specifically maintains VEX1 compartmentalisation following DNA replication. Thus, the VEX-complex controls VSG-exclusion, while CAF-1 sustains VEX-complex inheritance in association with the active-VSG. Notably, the VEX2-orthologue and CAF-1 in mammals are also implicated in exclusion and inheritance functions. In trypanosomes, these factors sustain a highly effective and paradigmatic immune evasion strategy.
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http://dx.doi.org/10.1038/s41467-019-10823-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6617441PMC
July 2019

The kinetochore and the origin of eukaryotic chromosome segregation.

Authors:
Mark C Field

Proc Natl Acad Sci U S A 2019 06 7;116(26):12596-12598. Epub 2019 Jun 7.

School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom;

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http://dx.doi.org/10.1073/pnas.1908067116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601246PMC
June 2019

Pore timing: the evolutionary origins of the nucleus and nuclear pore complex.

F1000Res 2019 3;8. Epub 2019 Apr 3.

The Rockefeller University, New York, USA.

The name "eukaryote" is derived from Greek, meaning "true kernel", and describes the domain of organisms whose cells have a nucleus. The nucleus is thus the defining feature of eukaryotes and distinguishes them from prokaryotes (Archaea and Bacteria), whose cells lack nuclei. Despite this, we discuss the intriguing possibility that organisms on the path from the first eukaryotic common ancestor to the last common ancestor of all eukaryotes did not possess a nucleus at all-at least not in a form we would recognize today-and that the nucleus in fact arrived relatively late in the evolution of eukaryotes. The clues to this alternative evolutionary path lie, most of all, in recent discoveries concerning the structure of the nuclear pore complex. We discuss the evidence for such a possibility and how this impacts our views of eukaryote origins and how eukaryotes have diversified subsequent to their last common ancestor.
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http://dx.doi.org/10.12688/f1000research.16402.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449795PMC
May 2020

Transcriptome, proteome and draft genome of Euglena gracilis.

BMC Biol 2019 02 7;17(1):11. Epub 2019 Feb 7.

School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.

Background: Photosynthetic euglenids are major contributors to fresh water ecosystems. Euglena gracilis in particular has noted metabolic flexibility, reflected by an ability to thrive in a range of harsh environments. E. gracilis has been a popular model organism and of considerable biotechnological interest, but the absence of a gene catalogue has hampered both basic research and translational efforts.

Results: We report a detailed transcriptome and partial genome for E. gracilis Z1. The nuclear genome is estimated to be around 500 Mb in size, and the transcriptome encodes over 36,000 proteins and the genome possesses less than 1% coding sequence. Annotation of coding sequences indicates a highly sophisticated endomembrane system, RNA processing mechanisms and nuclear genome contributions from several photosynthetic lineages. Multiple gene families, including likely signal transduction components, have been massively expanded. Alterations in protein abundance are controlled post-transcriptionally between light and dark conditions, surprisingly similar to trypanosomatids.

Conclusions: Our data provide evidence that a range of photosynthetic eukaryotes contributed to the Euglena nuclear genome, evidence in support of the 'shopping bag' hypothesis for plastid acquisition. We also suggest that euglenids possess unique regulatory mechanisms for achieving extreme adaptability, through mechanisms of paralog expansion and gene acquisition.
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http://dx.doi.org/10.1186/s12915-019-0626-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366073PMC
February 2019
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