Biology subjects

Jones, N. G.

Publications and source records attributed to Jones, N. G..

12 recordsLinked to original sources

bioRxiv2025

A non-canonical role for the ATAD2 ortholog BDF7 in nucleolar ribosome maturation and amastigote survival in Leishmania mexicana.

ATAD2 is a widely conserved, homohexameric ATPase that, in humans, features a bromodomain tier. It plays a role in chromatin remodelling in embryonic stem and germ cells but is frequently upregulated in many cancers, making its bromodomain an attractive drug target. While ATAD2-like proteins generally modulate nucleosome density to control genome compartmentalization and gene expression across eukaryotes, their precise molecular functions vary. We investigated LmxBDF7, the ATAD2 ortholog in the important human pathogen Leishmania mexicana, the causative agent of cutaneous leishmaniasis. Unlike its human counterpart, the LmxBDF7 bromodomain is predicted to be occluded and non-canonical. BDF7 null mutants were unable to develop into infectious amastigote forms and could not infect macrophages, demonstrating it is essential for lifecycle progression. Chromatin Immunoprecipitation sequencing (ChIP-seq) suggested low affinity for chromatin, aligning with its atypical bromodomain. Instead, Proximity-Biotinylation (XL-BioID) suggested a role in ribosome maturation within the nucleolus. RNA-sequencing (RNA-seq) of the {Delta}bdf7 mutant revealed widespread disruption of gene expression during growth and differentiation. Crucially, key genes required for amastigote survival, such as ribosomal protein genes and glutamine synthetase, were downregulated. This downregulation was spatially biased, preferentially affecting genes on Chromosome 23. Our combined data suggest that while BDF7 is essential, its functions have diverged from other ATAD2-like factors found in opisthokonts. Author SummaryATAD2 is a protein that helps cells balance the number of nucleosomes bound to DNA in the nucleus of a cell. Occurring at important sites or times this provides a helper function that ensures other protein complexes can operate on chromatin effectively. In some cancers ATAD2 is disrupted and therefore is being explored as target for new medicines. Orthologues of ATAD2 have been characterised in mammals and several species of yeast. We have sought to identify if an ATAD2-like protein can be found in the important human pathogen, Leishmania mexicana - which is evolutionary distant from humans and yeast. Indeed, we were able to find an ATAD2-like protein called BDF7. Interestingly, BDF7 has a bromodomain which is predicted to be non-functional in terms of being able to bind histones. We were able to make strains of Leishmania mexicana that lacked BDF7 which were viable, but unable to complete the differentiation step required for infecting macrophages. Intriguingly we found BDF7 had a poor association with chromatin and inhabited a protein neighbourhood defined by factors which facilitate ribosome biogenesis. Lastly, RNA-seq analysis of the cells revealed that those lacking BDF7 were potentially depleted for glutamine synthetase which would prevent them developing into fully functional amastigotes.

molecular biology↗

bioRxiv2025

Chemical genetics reveals Leishmania KKT2 and CRK9 kinase activity is required for cell cycle progression

Protein kinases are key regulators of the eukaryotic cell cycle and have consequently emerged as attractive targets for drug development. Their well-defined active sites make them particularly amenable to inhibition by small molecules, underscoring their druggability. The Leishmania kinome, shaped by diverse evolutionary processes, harbours a unique repertoire of potential drug targets. Here, we used the cysteine-directed protein kinase probe SM1-71 to identify four essential protein kinases MPK4, MPK5, MPK7 and AEK1 as candidates for covalent kinase inhibitor development, as well as CLK1/CLK2 for which covalent inhibitors have already been identified. We leveraged the absence of natural analog-sensitive (AS) kinases in L. mexicana to establish an in vivo chemical-genetic AS kinase platform for investigating essential functions of protein kinases. Using CRISPR-Cas9-mediated precision genome editing, we endogenously engineered two kinetochore-associated protein kinases, KKT2 and KKT3, and cyclin-dependent kinase CRK9, to generate AS kinases. We show that KKT2 and CRK9 kinase activities are essential for both promastigote and intracellular amastigote survival; KKT2 kinase activity being required for progression through mitosis at a stage preceding mitotic spindle assembly, while CRK9 kinase activity is required for S phase, consistent with its role in trans-splicing. This study demonstrates the utility of AS chemical genetics in Leishmania and identifies KKT2 and CRK9 as having critical roles in Leishmania cell cycle regulation and therefore being promising drug targets.

molecular biology↗

bioRxiv2025

Structure and Activity of the Essential UCH Family Deubiquitinase DUB16 from Leishmania donovani

In Leishmania parasites, as for their hosts, the ubiquitin proteasome system is important for cell viability. As part of a systematic gene deletion study, it was discovered that four cysteine protease type deubiquitinases (DUBs) are essential for parasite survival in the promastigote stage, including DUB16. Here we have purified and characterised recombinant DUB16 from Leishmania donovani, which belongs to the ubiquitin C-terminal hydrolase (UCH) family. DUB16 efficiently hydrolyses C-terminal aminocoumarin and rhodamine conjugates of ubiquitin consistent with proposed cellular roles of UCH-type DUBs in regenerating free monomeric ubiquitin from small molecule ubiquitin adducts arising from adventitious metabolic processes. The crystal structure of DUB16 reveals a typical UCH-type deubiquitinase fold, and a relatively short and disordered crossover loop that appears to restrict access to the catalytic cysteine. At close to stoichiometric enzyme to substrate ratios, DUB16 exhibits deubiquitinase activity towards diubiquitins linked through isopeptide bonds between Lys11, Lys48 or Lys63 residues of the proximal ubiquitin and the C-terminus of the distal ubiquitin. With 100-1000-fold higher turnover rates, DUB16 cleaves the ubiquitin-ribosomal L40 fusion protein to give the mature products. A DUB-targeting cysteine-reactive cyanopyrrolidine compound, IMP-1710, inhibits DUB16 activity. IMP-1710 was shown in promastigote cell viability assays to have parasite killing activity with EC50 values of 1-2 M, comparable to the anti-leishmanial drug, miltefosine. L. mexicana parasites engineered to overproduce DUB16 showed a modest increase in resistance to IMP-1710, providing evidence that IMP-1710 inhibits DUB16 in vivo. Together these results suggest on-target activity and that DUB16 may be a druggable target to develop new anti-leishmania compounds.

biochemistry↗

bioRxiv2023

The dual role of the 16mer motif within the 3' untranslated region of the variant surface glycoprotein of Trypanosoma brucei

The variant surface glycoprotein (VSG) of African trypanosomes is essential for survival of bloodstream form parasites. These parasites undergo antigenic variation, an immune evasion strategy in which they periodically switch VSG expression from one isoform to another. The molecular processes central to the expression and regulation of the VSG are however not fully understood. In general, the regulation of gene expression in trypanosomes is largely post-transcriptional. Regulatory sequences, mostly present in the 3 UTRs, often serve as key elements in the modulation of the levels of individual mRNAs. In T. brucei VSG genes, a 16mer motif within the 3 UTR has been shown to be essential for the stability of VSG transcripts and abundant VSG expression. This motif is 100 % conserved in the 3 UTRs of all transcribed and non-transcribed VSG genes. As a stability-associated sequence element, the absence of nucleotide substitutions in the 16mer is however exceptional. We therefore hypothesised that the motif is involved in other essential roles/processes besides stability of the VSG transcripts. In this study, we demonstrate that the 100 % conservation of the 16mer motif is not essential for cell viability or for the maintenance of functional VSG protein levels. We further show that the intact motif in the active VSG 3 UTR is neither required to promote VSG silencing during switching nor is it needed during differentiation from bloodstream forms to procyclic forms. Ectopic overexpression of a second VSG, however, requires the intact 16mer motif within the ectopic VSG 3 UTR to trigger silencing and exchange of the active VSG, suggesting a role for the motif in transcriptional VSG switching. The enigmatic 16mer motif therefore appears to play a dual role in transcriptional VSG switching and VSG transcript stability.

cell biology↗

bioRxiv2023

Bromodomain Factor 5 as a Target for Antileishmanial Drug Discovery

Leishmaniases are a collection of neglected tropical diseases caused by kinetoplastid parasites in the genus Leishmania. Current chemotherapies are severely limited and the need for new antileishmanials is of pressing international importance. Bromodomains are epigenetic reader domains that have shown promising therapeutic potential for cancer therapy and may also present an attractive target to treat parasitic diseases. Here, we investigate Leishmania donovani bromodomain factor 5 (LdBDF5) as a target for antileishmanial drug discovery. LdBDF5 contains pair of bromodomains (BD5.1 and BD5.2) in an N-terminal tandem repeat. We purified recombinant bromodomains of L. donovani BDF5 and determined the structure of BD5.2 by X-ray crystallography. Using a histone peptide microarray and fluorescence polarisation assay, we identified binding interactions of LdBDF5 bromodomains with acetylated peptides derived from histones H2B and H4. In orthogonal biophysical assays including thermal shift assays, fluorescence polarisation and NMR, we showed that BDF5 bromodomains bind to human bromodomain inhibitors SGC-CBP30, bromosporine and I-BRD9, moreover, SGC-CBP30 exhibited activity against Leishmania promastigotes in cell viability assays. These findings exemplify the potential BDF5 holds as a drug target in Leishmania and provide a foundation for the future development of optimised antileishmanial compounds targeting this epigenetic reader protein. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/554125v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@16be162org.highwire.dtl.DTLVardef@19a1cf3org.highwire.dtl.DTLVardef@e29c7forg.highwire.dtl.DTLVardef@190564d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes

Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early, late and recycling endosomes. To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for early, late, and recycling endosomes. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.

cell biology↗

bioRxiv2021

Bromodomain factor 5 is an essential transcriptional regulator of the Leishmania genome.

Leishmania are unicellular parasites that cause human and animal disease. Alongside other organisms in kinetoplastida, they have evolved an unusual genome architecture that requires all RNA polymerase II transcribed genes to be expressed constitutively, with transcriptional start regions denoted by histone variants and histone lysine acetylation. However, the way these chromatin marks are interpreted by the cell is not understood. Seven predicted bromodomain factors (BDF1-7), the reader modules for acetyl-lysine, were identified across Leishmania genomes. Using L. mexicana as a model, Cas9-driven gene deletions indicate that BDF1-5 are essential for promastigote survival, whilst DiCre inducible gene deletion of the dual bromodomain factor BDF5 identified it to be essential for both promastigotes and amastigotes. ChIP-seq assessment of BDF5s genomic distribution revealed it as highly enriched at transcriptional start sites. Using an optimised proximity proteomic and phosphoproteomic technique, XL-BioID, we defined the BDF5-proximal environment to be enriched for other bromodomain factors, histone acetyltransferase 2, and proteins essential for transcriptional activity and RNA processing. Inducible deletion of BDF5, led to a disruption of pol II transcriptional activity and global defects in gene expression. Our results indicate the requirement of Leishmania to interpret histone acetylation marks for normal levels of gene expression and thus cellular viability.

cell biology↗

bioRxiv2021

Protein kinase signalling at the Leishmania kinetochore captured by XL-BioID

Elucidating protein kinase signaling pathways is an important but challenging problem in cell biology. Phosphoproteomics has been used to identify many phosphorylation sites, however the spatial context of these sites within the cell is mostly unknown, making it difficult to reconstruct signalling pathways. To address this problem an in vivo proximity capturing workflow was developed, consisting of proximity biotinylation followed by protein cross-linking (XL-BioID). This was applied to protein kinases of the Leishmania kinetochore, leading to the discovery of a novel essential kinetochore protein, KKT26. XL-BioID enabled the quantification of proximal phosphosites at the kinetochore through the cell cycle, allowing the phosphorylation state of the kinetochore to be followed during assembly. A specific inhibitor of kinetochore protein kinases KKT10/KKT19 was used to show that XL-BioID provides a spatially focussed view of protein kinase inhibition, identifying 16 inhibitor-responsive proximal phosphosites, including 3 on KKT2, demonstrating the potential of this approach for discovery of in vivo kinase signalling pathways.

biochemistry↗

bioRxiv2021

The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei

The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.

microbiology↗

bioRxiv2021

Reduced calcium storage blunts calcium signaling in Toxoplasma bradyzoites and impedes motility and egress

Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e. tachyzoites) and for establishing chronic infection (i.e. bradyzoites). Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unexplored. Here we show that Ca2+ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened calcium responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with down-regulation of calcium ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus calcium rapidly restored their intracellular Ca2+ and ATP stores leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca2+ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility.

microbiology↗

bioRxiv2021

Endoplasmic reticulum-targeting but not translation is required for mRNA balancing in trypanosomes

The cell surface of bloodstream form African trypanosomes is covered by a dense coat of immunogenic variant surface glycoproteins (VSGs). By continuously changing the expressed VSG antigen, the parasites can survive the hosts immune response. The VSG is highly expressed in Trypanosoma brucei, accounting for approximately 10 - 20% of total mRNA. Depletion of VSG mRNA is lethal, and a counterbalancing of the mRNA levels occurs when two or more VSGs are simultaneously expressed. How the VSG expression levels are regulated is unknown. Here, by using inducible and constitutive systems for ectopic VSG expression, we have discovered that (i) the endogenous VSG mRNA level is downregulated only when the ectopic VSG is targeted to the ER, (ii) VSG translation is dispensable and in fact, (iii) the regulation of VSG mRNA levels does not depend on a VSG open reading frame. We propose that feedback elicited at the ER regulates the VSG mRNA amounts to avoid overshooting the secretory pathway capacity. In this way, VSG expression is quantitatively and qualitatively fine-tuned. Balancing the overall number of ER-targeted mRNAs could well be a general mechanism in cell biology. The trypanosome system with just one dominant mRNA species provides a versatile model for studying this phenomenon.

microbiology↗

bioRxiv2021

Nanobody Mediated Macromolecular Crowding Induces Membrane Fission and Remodeling in the African Trypanosome

The dense Variant Surface Glycoprotein (VSG) coat of African trypanosomes represents the primary host-pathogen interface. Antigenic variation prevents clearing of the pathogen by employing a large repertoire of antigenically distinct VSG genes, thus neutralizing the hosts antibody response. To explore the epitope space of VSGs, we generated anti-VSG nanobodies and combined high-resolution structural analysis of VSG-nanobody complexes with binding assays on living cells, revealing that these camelid antibodies bind deeply inside the coat. One nanobody caused rapid loss of cellular motility, possibly due to blockage of VSG mobility on the coat, whose rapid endo-and exocytosis is mechanistically linked to T. brucei propulsion and whose density is required for survival. Electron microscopy studies demonstrated that this loss of motility was accompanied by rapid formation and shedding of nanovesicles and nanotubes, suggesting that increased protein crowding on the dense membrane can be a driving force for membrane fission in living cells.

microbiology↗