Biology subjects

Field, M. C.

Publications and source records attributed to Field, M. C..

9 recordsLinked to original sources

bioRxiv2025

A Ras-like GTPase in African trypanosomes

Small Ras-like GTPases are conserved across eukaryotes and mediate many functions. Trypanosoma brucei encodes only three putative open reading frames belonging to Ras and Rho subfamilies, all highly divergent from other taxa. We investigated the most Ras-like gene product in T. brucei, TbRlp, evaluating essentiality, location and impact of blocking the GDP/GTP cycle. TbRlp is expressed in both life stages, with a clear fitness contribution in bloodstream forms in in vitro culture, without a specific block to the cell cycle or clear morphological defect. Overexpression of TbRlp and mutant forms results in moderate disruption of the cell cycle and altered proliferation indicating that GTP/GDP cycling and expression levels of these forms of TbRlp are important to proliferation. Epitope-tagged TbRlp is located predominantly on structures in the posterior of the cell, and suggesting the late endosomal system. Analyses also indicates a link between cell density and levels of TbRlp in the BSF, with a decrease in TbRlp protein but not RNA upon increased cell density. These data suggest that TbRlp is important in control of proliferation and opens possibilities for future research.

cell biology↗

bioRxiv2024

Targeted protein degradation of the CPSF complex by benzoxaboroles through sumoylation

Benzoxaboroles (BoBs) feature a boron-heterocyclic core and are an important 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 (MoA) 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. Here we establish a functional connection between protein sumoylation and the boron-heterocyclic scaffold shared by all BoBs using comprehensive genetic screens in Trypanosoma brucei. There is a rapid temporal and spatial shift in global protein sumoylation following BoB exposure and members of the CPSF complex are specifically destabilised in a SUMO and proteosome-dependent manner. Finally, we find rapid decrease in bulk mRNA levels, consistent with the role of CPSF3 in mRNA maturation. We propose that a combination of direct inhibition coupled with targeted degradation of CPSF3 underpins the specificity of BoBs against trypanosomatids.

cell biology↗

bioRxiv2023

Trypanosoma cruzi VDU deubiquitinase mediates surface protein trafficking and infectivity

Ubiquitylation is a post-translational modification promoting protein degradation. Within the endomembrane system ubiquitylation marks proteins for lysosomal processing. Deubiquitinases (DUBs) cleave ubiquitin from modified proteins and, in the case of surface proteins, prevent lysosomal targeting and thus play a major role in controlling turnover. In Trypanosoma cruzi, the etiological agent of Chagas disease, acquisition of nutrients in parasites proliferating within the blood meal of insect vectors occurs via the cytostome, a unique structure connected to a long tubular cytopharynx. Contents are delivered to late endosomes and accumulate in reservosomes, equivalent to lysosomes. When starved, T. cruzi differentiates into mammalian infective trypomastigotes, which are cell cycle arrested. Here we asked what roles ubiquitylation plays in this unique endocytic process by interrogation of the T. cruzi ortholog of VDU (von Hippel-Lindau-interacting deubiquitylating enzyme)/USP33 (ubiquitin-specific protease). We found that TcVDU expression level inversely correlated with transferrin endocytosis, and that overexpression led to a longer retention of the endocytic cargo near the cytostome. TcVDU itself was found enriched in the anterior region of the parasite, in proximity to endocytic cargo. Most importantly, TcVDU overexpression reduced parasite invasion capacity and led to increased release of trans-sialidase. These alterations in the abundance of multiple surface proteins in TcVDU mutants indicate a key role of TcVDU in modulating the T. cruzi surface by affecting the endosomal traffic and consequently the host-parasite interface. Author summaryTrypanosoma cruzi is the cause of Chagas disease that affects large populations of Central and South America. Disease is spreading to other continents due to poor control of blood transfusion from donors with chronic and undiagnosed infection, migration and drug treatment with limited performance and undesired side effects. Proliferating forms of T. cruzi acquire nutrients through the cytostome, a cell surface opening connected to a long tubular cytopharynx. Internalized material is endocytosed in the cytopharynx. This is distinct from other members of the Trypanosomatids, which endocytose material exclusively through the flagellar pocket. By using CRISPR gene editing and overexpression we demonstrated that a conserved deubiquitinase (VDU) is a key control element for traffic from the cytopharynx to endosomal compartments in T. cruzi. Changed levels of VDU modify endosomal traffic and largely impact the parasite surface, causing changes in infectivity.

microbiology↗

bioRxiv2022

Functional differentiation of Sec13 paralogs in the euglenozoan protists

The {beta}-propeller protein Sec13 plays roles in at least three distinct processes by virtue of being a component of the COPII endoplasmic reticulum export vesicle coat, the nuclear pore complex (NPC) and the Seh1-associated (SEA)/GATOR nutrient-sensing complex, suggesting that regulatory mechanisms coordinating these cellular activities may operate via Sec13. The NPC, COPII and SEA/GATOR are all ancient features of eukaryotic cells. In the vast majority of eukaryotes, a single Sec13 gene is present, but here we report that the Euglenozoa, a lineage encompassing the diplonemid, kinetoplastid and euglenid protists contain two Sec13 paralogs. Furthermore, based on protein interactions and localization studies we show that Sec13 functions in diplonemids are divided between the Sec13a and Sec13b paralogs. Specifically, Sec13a interacts with COPII and the NPC, but Sec13b interacts with Sec16 and components of the SEA/GATOR complex. We infer that euglenozoan Sec13a is responsible for NPC functions and canonical anterograde transport activities while Sec13b acts within nutrient and autophagy-related pathways, indicating a fundamentally distinct organization of coatomer complexes in the euglenozoan flagellates.

evolutionary biology↗

bioRxiv2022

Mex67 paralogs mediate division of labor in trypanosome RNA processing and export.

In opistokhonts (animals and fungi), mRNA export to the cytoplasm is mediated by the Mex67/Mtr2 (NXF1/NXT1) heterodimer via the nuclear pore complex (NPC). In contrast to most nucleocytoplasmic transport, mRNA export requires ATP-dependent remodeling machinery, and in animals and fungi is Ran-independent. While most eukaryotes possess one Mex67 gene, trypanosomes have three distinct Mex67 paralogs, while retaining a single Mtr2 gene. We show here that these paralogs, TbMex67, TbMex67b and TbMex67L, have differing and non-redundant roles in RNA export. Specifically, TbMex67 and TbMex67b retain a canonical role in mRNA export, albeit associating with specific mRNA cohorts, but in contrast, TbMex67L is primarily involved in ribosome biogenesis. Together with the association of all Mex67 paralogs with the Ran machinery, these findings indicate significant departures in RNA export mechanisms in these divergent organisms, with implications for evolutionary origins and diversity in control of gene expression.

cell biology↗

bioRxiv2020

The endosomal TbTpr86/TbUsp7/SkpZ (TUS) complex controls surface protein abundance in trypanosomes

In trypanosomes the orthologs of human USP7 and VDU1 control abundance of a cohort of surface proteins, including invariant surface glycoproteins (ISGs) by functioning as deubiquitinases (DUBs) Silencing TbUsp7 partially inhibits endocytosis and invariant surface glycoprotein turnover. As a component of cullin E3 ubiquitin ligases, 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. 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 and 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 TbTpr86, TbUsp7 and SkpZ form the TUS complex that regulates abundance of a significant cohort of trypanosome surface proteins.

cell biology↗

bioRxiv2020

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

Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. African trypanosome pentamidine and melarsoprol sensitivity 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 or via binding to TbAQP2, with subsequent endocytosis, but trafficking and regulation of TbAQPs is uncharacterised. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmic lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation also leads to impaired oligomerisation, mislocalisation, and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and contributes towards emergence of drug resistance.

microbiology↗

bioRxiv2019

Modification of an atypical clathrin-independent AP-2 adaptin complex of Plasmodium falciparum reduces susceptibility to artemisinin

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin. In the Mekong region this is associated with mutations in the kelch propeller-encoding domain of pfkelch13, but variants of other parasite proteins are also thought to modulate the response to drug. Evidence from human and rodent studies suggests that the -subunit of the AP-2 adaptin trafficking complex is one such protein of interest. We generated transgenic Plasmodium falciparum parasites encoding the I592T variant of pfap2, orthologous to the I568T mutation associated with in vivo artemisinin resistance in P. chabaudi. When exposed to a four-hour pulse of dihydroartemisin in the ring-stage survival assay, two P. falciparum clones expressing AP-2 I592T displayed significant and reproducible survival of 8.0% and 10.3%, respectively, compared to <2% for the 3D7 parental line (P = 0.0011 for each clone). In immunoprecipitation and localisation studies of HA-tagged AP-2, we identified interacting partners including AP-2, AP-1/2{beta}, AP-2{sigma} and a kelch-domain protein encoded on chromosome 10 of P. falciparum, K10. Conditional knockout indicates that the AP-2 trafficking complex in P. falciparum is essential for the fidelity of merozoite biogenesis and membrane organisation in the mature schizont. We also show that while other heterotetrameric AP-complexes and secretory factors interact with clathrin, AP-2 complex subunits do not. Thus, the AP-2 complex may be diverted from a clathrin-dependent endocytic role seen in most eukaryotes into a Plasmodium-specific function. These findings represent striking divergences from eukaryotic dogma and support a role for intracellular traffic in determining artemisinin sensitivity in vitro, confirming the existence of multiple functional routes to reduced ring-stage artemisinin susceptibility. Therefore, the utility of pfkelch13 variants as resistance markers is unlikely to be universal, and phenotypic surveillance of parasite susceptibility in vivo may be needed to identify threats to our current combination therapies.

microbiology↗

bioRxiv2019

Proteome of the secondary plastid of Euglena gracilis reveals metabolic quirks and colourful history

Euglena gracilis is a well-studied biotechnologically exploitable phototrophic flagellate harbouring secondary green plastids. Here we describe its plastid proteome obtained by high-resolution proteomics. We identified 1,345 candidate plastid proteins and assigned functional annotations to 774 of them. More than 120 proteins are affiliated neither to the host lineage nor the plastid ancestor and may represent horizontal acquisitions from various algal and prokaryotic groups. Reconstruction of plastid metabolism confirms both the presence of previously studied/predicted enzymes/pathways and also provides direct evidence for unusual features of its metabolism including uncoupling of carotenoid and phytol metabolism, a limited role in amino acid metabolism and the presence of two sets of the SUF pathway for FeS cluster assembly. Most significantly, one of these was acquired by lateral gene transfer (LGT) from the chlamydiae. Plastidial paralogs of membrane trafficking-associated proteins likely mediating a poorly understood fusion of transport vesicles with the outermost plastid membrane were identified, as well as derlin-related proteins that potentially act as protein translocases of the middle membrane, supporting an extremely simplified TIC complex. The proposed innovations may be also linked to specific features of the transit peptide-like regions described here. Hence the Euglena plastid is demonstrated to be a product of several genomes and to combine novel and conserved metabolism and transport processes.

evolutionary biology↗