Biology subjects

Gas-Pascual, E.

Publications and source records attributed to Gas-Pascual, E..

7 recordsLinked to original sources

bioRxiv2026

Potential for the Terminal SKP1 Glycosyltransferase to Exert Non-Enzymatic Control of SKP1 in Toxoplasma gondii

The SKP1/CUL1/F-Box protein (SCF) family of E3 ubiquitin ligases is responsible for targeting multiple proteins for degradation by the 26S proteosome. Within the family, multiple F-box proteins (FBPs) associate with the SKP1 adaptor which in turn docks to CUL1. In the human parasite Toxoplasma gondii, SKP1 is subject to oxygen-dependent regulation. Under normoxic conditions, the prolyl hydroxylase PHYa hydroxylates SKP1 priming it for modification by five SKP1-specific glycosyltransferase activities. Glycosylation weakens competing homodimerization of SKP1, and because SKP1 is limiting in cells, this affects the profile of bound FBPs. A previous study noted that the terminal SKP1 glycosyltransferase, GAT1, is a stable member of the SKP1 interactome regardless of SKP1s glycosylation status. Furthermore, gat1-knockout cells exhibit a unique repertoire of FBPs bound to SKP1 relative to normal and other glycosylation-defective mutants. Utilizing sedimentation velocity analytical ultracentrifugation, we demonstrate that the native GAT1 homodimer complexes with SKP1 monomers with affinity and stoichiometry dictated by its glycostate. Computational modeling validated by mutational probing shows that GAT1 competes with the core hydrophobic interface also utilized by FBPs and the SKP1 homodimer. This interface is complemented by varying, transient fuzzy-like interactions contributed by SKP1s intrinsically disordered C-terminal region (CTR) that are in turn constrained by the glycan. Furthermore, GAT1 substoichiometrically monomerizes SKP1 in a CTR-dependent manner. Thus, GAT1 is specific for monomeric SKP1, and the mechanism by which this is achieved also modifies the dimer/monomer equilibrium of unmodified SKP1 with downstream effects on the steady-state profile of FBP/SKP1 complexes in cells.

biochemistry↗

bioRxiv2025

Global characterization of Dictyostelium discoideum gene expression changes under hypoxic conditions

Aerobic eukaryotes utilize O2 to oxidize metabolites and generate ATP. The protist kingdom lacks the Hypoxia Inducible Factor-dependent transcriptional response network to accommodate low O2 though possesses a HIF prolyl hydroxylase that initially mediates the response. To address the scale and scope of hypoxic responses in protists, we characterized transcriptomic and proteomic changes when the social amoeba Dictyostelium is subjected to low (1%) O2 under nutritive conditions over 24 h followed by reoxygenation. Remarkably, 32% of the transcripts quantified were differentially expressed during hypoxia, with greatest changes associated with early (1 h) and late phases (24 h). Protein changes were modestly correlated with and generally lagged behind transcriptional changes. Correlated changes were observed for transcripts and proteins associated with various metabolic, anabolic, and catabolic pathways, as well as chromosome organization, cell cycling, vesicular trafficking, and signaling. Analysis of 4 marker genes showed extremely rapid responses that were graded over a range of O2 levels, with differential responses to inhibitors affecting protein synthesis and mitochondria. Overall, the amoebal response to a low but non-toxic O2-level resulted in massive remodeling of the transcriptome and proteome.

genomics↗

bioRxiv2024

Novel antibodies detect nucleocytoplasmic O-fucose in protist pathogens, cellular slime molds, and plants

Cellular adaptations to change often involve post-translational modifications of nuclear and cytoplasmic proteins. An example found in protists and plants is the modification of serine and threonine residues of dozens to hundreds of nucleocytoplasmic proteins with a single fucose (O-Fuc). A nucleocytoplasmic O-fucosyltransferase (OFT) occurs in the pathogen Toxoplasma gondii, the social amoeba Dictyostelium, and higher plants, where it is called Spy because mutants have a spindly appearance. O-fucosylation, which is required for optimal proliferation of Toxoplasma and Dictyostelium, is paralogous to the O-GlcNAcylation of nucleocytoplasmic proteins of plants and animals that is involved in stress and nutritional responses. O-Fuc was first discovered in Toxoplasma using Aleuria aurantia lectin, but its broad specificity for terminal fucose residues on N- and O-linked glycans in the secretory pathway limits its use. Here we present affinity purified rabbit antisera that are selective for the detection and enrichment of proteins bearing fucose-O-Ser or fucose-O-Thr. These antibodies detect numerous nucleocytoplasmic proteins in Toxoplasma, Dictyostelium, and Arabidopsis, as well as O-Fuc occurring on secretory proteins of Dictyostelium and mammalian cells, although the latter are frequently blocked by further glycosylation. The antibodies label Toxoplasma, Acanthamoeba, and Dictyostelium in a pattern reminiscent of O-GlcNAc in animal cells including nuclear pores. The O-fucome of Dictyostelium is partially conserved with that of Toxoplasma and is highly induced during starvation-induced development. These antisera demonstrate the unique antigenicity of O-Fuc, document conservation of the O-fucome among unrelated protists, and will enable the study of the O-fucomes of other organisms possessing OFT-like genes. IMPORTANCEO-fucose, a form of mono-glycosylation on serine and threonine residues of nuclear and cytoplasmic proteins of some parasites, other unicellular eukaryotes, and plants, is understudied because it is difficult to detect owing to its neutral charge and lability during mass spectrometry. Yet the O-fucosyltransferase enzyme (OFT) is required for optimal growth of the agent for toxoplasmosis, Toxoplasma gondii, and an unrelated protist, the social amoeba Dictyostelium discoideum. Furthermore, O-fucosylation is closely related to the analogous process of O-GlcNAcylation of thousands of proteins of animal cells, where it plays a central role in stress and nutritional responses. O-Fuc is currently best detected using Aleuria aurantia lectin (AAL), but in most organisms AAL also recognizes a multitude of proteins in the secretory pathway that are modified with fucose in different ways. By establishing the potential to induce highly specific rabbit antisera that discriminate O-Fuc from all other forms of protein fucosylation, this study expands knowledge about the protist O-fucome and opens a gateway to explore the potential occurrence and roles of this intriguing posttranslational modification in bacteria and other protist pathogens such as Acanthamoeba castellanii.

cell biology↗

bioRxiv2024

The GPI sidechain of Toxoplasma gondii prevents parasite pathogenesis

Glycosylphosphatidylinositols (GPIs) are highly conserved anchors for eukaryotic cell surface proteins. The apicomplexan parasite, Toxoplasma gondii, is a widespread intracellular parasite of warm-blooded animals whose plasma membrane is covered with GPI-anchored proteins, and free GPIs called GIPLs. While the glycan portion is conserved, species differ in sidechains added to the triple mannose core. The functional significance of the Glc1,4GalNAc{beta}1-sidechain reported in Toxoplasma gondii has remained largely unknown without an understanding of its biosynthesis. Here we identify and disrupt two glycosyltransferase genes and confirm their respective roles by serology and mass spectrometry. Parasites lacking the sidechain on account of deletion of the first glycosyltransferase, PIGJ, exhibit increased virulence during primary and secondary infections, suggesting it is an important pathogenesis factor. Cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants are intact. In contrast, the scavenger receptor CD36 shows enhanced binding to PIGJ mutants, potentially explaining a subtle tropism for macrophages detected early in infection. Galectin-3, which bind GIPLs, exhibits a slight enhancement of binding to PIGJ mutants, and the protection of galectin-3 knockout mice from lethality suggests that{Delta} pigj parasite virulence in this context is sidechain dependent. Parasite numbers are not affected by{Delta} pigj early in the infection in wildtype mice, suggesting a breakdown of tolerance. However, increased tissue cysts in the brains of mice infected with{Delta} pigj parasites indicate an advantage over wildtype strains. Thus, the GPI sidechain of T. gondii plays a crucial and diverse role in regulating disease outcome in the infected host. SummaryThe functional significance of sidechain modifications to the GPI anchor is yet to be determined because the glycosyltransferases responsible for these modifications have not been identified. Here we present identification and characterization of both T. gondii GPI sidechain-modifying glycosyltransferases. Removal of the glycosyltransferase that adds the first GalNAc to the sidechain results in parasites without a sidechain on the GPI, and increased parasite virulence. Loss of the second glycosyltransferase results in a sidechain with GalNAc alone, and no glucose added, and has negligible effect on parasite virulence. This indicates GPI sidechains as fundamental to host-parasite interactions.

microbiology↗

bioRxiv2023

Toxoplasma gondii F-Box Protein L2 Silences Feline-Restricted Genes Necessary for Sexual Commitment

Toxoplasma gondii is a foodborne pathogen that can cause severe and life-threatening infections in fetuses and immunocompromised patients. Felids are its only definitive hosts, and a wide range of animals, including humans, serve as intermediate hosts. When the transmissible bradyzoite stage is orally ingested by felids, they transform into merozoites that expand asexually, ultimately generating millions of gametes for the parasite sexual cycle. However, bradyzoites in intermediate hosts differentiate exclusively to disease-causing tachyzoites, which rapidly disseminate throughout the host. Though tachyzoites are well-studied, the molecular mechanisms governing transitioning between developmental stages are poorly understood. Each parasite stage can be distinguished by a characteristic transcriptional signature, with one signature being repressed during the other stages. Switching between stages requires substantial changes in the proteome, which is achieved in part by ubiquitination. F-box proteins mediate protein poly-ubiquitination by recruiting substrates to SKP1, Cullin-1, F-Box protein E3 ubiquitin ligase (SCF-E3) complexes. We have identified an F-box protein named Toxoplasma gondii F-Box Protein L2 (TgFBXL2), which localizes to distinct nuclear sites. TgFBXL2 is stably engaged in an SCF-E3 complex that is surprisingly also associated with a COP9 signalosome complex that negatively regulates SCF-E3 function. At the cellular level, TgFBXL2-depleted parasites are severely defective in centrosome replication and daughter cell development. Most remarkable, RNA seq data show that TgFBXL2 conditional depletion induces the expression of genes necessary for sexual commitment. We suggest that TgFBXL2 is a latent guardian of sexual stage development in Toxoplasma and poised to remove conflicting proteins in response to an unknown trigger of sexual development. AUTHOR SUMMARYToxoplasma gondii is a protozoan parasite that replicates sexually in felids and asexually in nearly all other mammals with each life stage having a specific transcriptional profile. When life stage specific transcription is not properly controlled, the parasite dies and therefore its important to understand what inhibits expression of sexual stage genes during asexual growth and vice versa. Here we identify a ubiquitin E3 ligase complex that inhibits sexual stage gene expression during asexual growth.

microbiology↗

bioRxiv2019

A glycogenin homolog controls Toxoplasma gondii growth via glycosylation of an E3 ubiquitin ligase

Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is uniquely modified in protists by an O2-dependent prolyl hydroxylase that generates the attachment site for a defined pentasaccharide. Previous studies demonstrated the importance of the core glycan for growth of the parasite Toxoplasma gondii in fibroblasts, but the significance of the non-reducing terminal sugar was unknown. Here, we find that a homolog of glycogenin, an enzyme that can initiate and prime glycogen synthesis in yeast and animals, is required to catalyze the addition of an -galactose in 3-linkage to the subterminal glucose to complete pentasaccharide assembly in cells. A strong selectivity of the enzyme (Gat1) for Skp1 in extracts is consistent with other evidence that Skp1 is the sole target of the glycosyltransferase pathway. gat1-disruption results in slow growth attesting to the importance of the terminal sugar. Molecular dynamics simulations provide an explanation for this finding and confirm the potential of the full glycan to control Skp1 organization as in the amoebozoan Dictyostelium despite the different terminal disaccharide assembled by different glycosyltransferases. Though Gat1 also exhibits low -glucosyltransferase activity like glycogenin, autoglycosylation is not detected and gat1-disruption reveals no effect on starch accumulation. A crystal structure of the ortholog from the crop pathogen Pythium ultimum explains the distinct substrate preference and regiospecificity relative to glycogenin. A phylogenetic analysis suggests that Gat1 is related to the evolutionary progenitor of glycogenin, and acquired a role in glycogen formation following the ancestral disappearance of the underlying Skp1 glycosyltransferase prior to amoebozoan emergence.

microbiology↗

bioRxiv2018

Enabling tools for Toxoplasma glycobiology

Infection by the protozoan parasite Toxoplasma gondii is a major health risk owing to its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Like nearly all eukaryotes, Toxoplasma glycosylates many of its proteins and lipids and assembles polysaccharides. Unlike most eukaryotes, Toxoplasma divides and differentiates in vacuoles within host cells. While their glycans resemble canonical models, they exhibit species-specific variations that have inhibited deeper investigations into their roles in parasite biology and virulence. The genome of Toxoplasma encodes a suite of likely glycogenes expected to assemble a range of N-glycans, O-glycans, a C-glycan, GPI-anchors, and polysaccharides, along with their requisite precursors and membrane transporters. To facilitate genetic approaches to investigate the roles of specific glycans, we mapped probable connections between 59 glycogenes, their enzyme products, and the glycans to which they contribute. We adapted a double-CRISPR/Cas9 strategy and a mass spectrometry-based glycomics workflow to test these relationships, and conducted infection studies in fibroblast monolayers to probe cellular functions. Through the validated disruption of 17 glycogenes, we also discovered novel Glc0-2-Man6-GlcNAc2-type N-glycans, GalNAc2- and Glc-Fuc-type O-glycans, and a nuclear O-Fuc type glycan. We describe the guide sequences, disruption constructs, and mutant strains, which are freely available to practitioners for application in the context of the relational map to investigate the roles of glycans in their favorite biological processes.

microbiology↗