The Skp1 Protein from Toxoplasma Is Modified by a Cytoplasmic Prolyl 4-Hydroxylase Associated with Oxygen Sensing in the Social Amoeba Dictyostelium

Xu, Yuechi; Brown, Kevin M.; Wang, Zhuo A.; Wel, Hanke van der; Teygong, Crystal; Zhang, Dongmei; Blader, Ira J.; West, Christopher M.

doi:10.1074/jbc.m112.355446

Cited by 49 publications

(81 citation statements)

References 43 publications

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“…In the host and presumably in the parasite, the donor for the OGT reaction, UDP-GlcNAc, is sensitive to the metabolic state of the cell, and modification by O-GlcNAc affects protein activity (36). Similarly, glycosylation of Skp1 is required for normal growth of tachyzoites in culture, and proline hydroxylation on Skp1 is sensitive to the redox status of T. gondii (22,29). In contrast, this study suggests that the addition of O-Fuc targets T. gondii proteins to assemblies closely associated with the nuclear membrane and that targeting of endogenous and exogenous proteins to the AAL-labeled assembly may occur in the absence of an NLS.…”

Section: Discussionmentioning

confidence: 99%

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O -fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii

Bandini

Haserick

Motari

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Toxoplasma gondii is an intracellular parasite that causes disseminated infections in fetuses and immunocompromised individuals. Although gene regulation is important for parasite differentiation and pathogenesis, little is known about protein organization in the nucleus. Here we show that the fucose-binding Aleuria aurantia lectin (AAL) binds to numerous punctate structures in the nuclei of tachyzoites, bradyzoites, and sporozoites but not oocysts. AAL also binds to Hammondia and Neospora nuclei but not to more distantly related apicomplexans. Analyses of the AAL-enriched fraction indicate that AAL binds O-linked fucose added to Ser/Thr residues present in or adjacent to Ser-rich domains (SRDs). Sixty-nine Ser-rich proteins were reproducibly enriched with AAL, including nucleoporins, mRNA-processing enzymes, and cell-signaling proteins. Two endogenous SRDs-containing proteins and an SRD-YFP fusion localize with AAL to the nuclear membrane. Superresolution microscopy showed that the majority of the AAL signal localizes in proximity to nuclear pore complexes. Host cells modify secreted proteins with O-fucose; here we describe the O-fucosylation pathway in the nucleocytosol of a eukaryote. Furthermore, these results suggest O-fucosylation is a mechanism by which proteins involved in gene expression accumulate near the NPC.toxoplasma | fucose | nuclear glycosylation | nuclear pore complex T he apicomplexan parasite Toxoplasma gondii causes disseminated infections in humans, and these infections can lead to severe damage in immunocompromised individuals and fetuses (1, 2). There is no human vaccine against T. gondii, and recently the price of pyrimethamine, the drug used to treat toxoplasmosis in the United States, has increased more than 50-fold (2).T. gondii has a complex life cycle, and the parasite's ability to differentiate through its life stages in response to stresses and environmental conditions is fundamental for its pathogenicity and transmission (3). Transcriptome analyses have revealed that a large percentage of mRNAs show life stage-specific expression (4) and/or cell cycle regulation (5). Recent studies have increased our understanding of gene expression in T. gondii by identifying the AP2 family of transcription factors (6-8) and by describing posttranslational modifications (PTMs) of histones and some of the enzymes responsible for them (9-11). However, little is known about protein organization at the nuclear periphery, a subnuclear compartment that plays a critical role in transcriptional regulation in many eukaryotes. In particular, the gene-gating model (12) suggests that the nuclear pore complex (NPC) has a role in transcriptional regulation and chromatin organization as well as in protein and mRNA transport (13,14).In T. gondii chromodomain protein 1 localizes with heterochromatin at the nuclear periphery (15), and centromeres sequester to an apical nuclear region (16). Although the nuclear localization signal (NLS) and importin-α system are present, key nuclear import and export molecules are...

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Section: Discussionmentioning

confidence: 99%

“…T. gondii is one of a few organisms that add a pentasaccharide containing GlcNAc, Gal, and fucose to a hydroxyl-proline on Skp1, a E3 ubiquitin ligase adaptor (22,29). Knockout of Skp1 proline hydroxylase (phyA), GlcNAc transferase (gnt1), and bifunctional galactose and fucose transferase (pgtA) did not affect AAL binding (Fig.…”

Section: Significancementioning

confidence: 99%

O -fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii

Bandini

Haserick

Motari

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The resulting plasmid was digested with XbaI and NotI and ligated to the XbaI-and NotI-digested 3Ј-flank. The resulting vector was linearized with KpnI and electroporated into RH⌬⌬ strain as described (5). Drug-resistant transformants were selected in the presence of 25 g/ml MPA and 25 g/ml xanthine and cloned by limiting dilution.…”

Section: Methodsmentioning

confidence: 99%

“…Western Blotting-Western blotting was performed as described (5). Briefly, tachyzoite pellets were solubilized in lysis buffer containing 8 M urea, 50 mM HEPES-NaOH, pH 7.4, supplemented with protease inhibitors at 4°C for 30 min and centrifuged at 16,000 ϫ g for 15 min at 4°C to generate a soluble S16 fraction.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, disruption of the gene for PhyA, the prolyl 4-hydroxylase that hydroxylates Pro-154 in Skp1, was observed to reduce tachyzoite proliferation in cell culture and fitness in a competition assay (5). Skp1 is an adaptor in the Skp1/Cullin-1/ F-box protein (SCF) 2 class of E3 ubiquitin ligases, and its hydroxylation was hypothesized to contribute to O 2 -dependent proliferation.…”

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The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development

Rahman

Zhao

Mandalasi

et al. 2016

Journal of Biological Chemistry

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Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIF␣ prolyl hydroxylases is required for optimal parasite proliferation, especially at low O 2 . We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O 2 -dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.Toxoplasma is a worldwide obligate intracellular apicomplexan parasite that infects most nucleated cells of warm-blooded animals (1). Toxoplasmosis, the disease caused by Toxoplasma, is an opportunistic infection in AIDS and other immune-suppressed patients (2). In addition, in utero infections can cause mental retardation, blindness, and death (3). Toxoplasma is transmitted by digesting parasites from feline feces (as oocysts) or undercooked meat (as tissue cysts). Once in the host, parasites convert to the tachyzoite form that disseminates to peripheral tissues (e.g. brain, retina, and muscle). The resulting immune response and/or drugs can control tachyzoite replication, but the parasite survives by converting into slow growing bradyzoites that encyst. Cysts sporadically burst, and the released parasites convert to tachyzoites whose unabated growth, as can occur in immune suppressed hosts, results in cell and tissue damage (4). Currently, no Toxoplasma vaccine exists; anti-toxoplasmosis drugs have severe side effects, and resistance to these drugs is occurring.Recently, disruption of the gene for PhyA, the prolyl 4-hydroxylase that hydroxylates Pro-154 in Skp1, was observed to reduce tachyzoite proliferation in cell culture and fitness in a competition assay (5). Skp1 is an adaptor in the Skp1/Cullin-1/ F-box protein (SCF) 2 class of E3 ubiq...

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Adventures in Defining Roles of Oxygenases in the Regulation of Protein Biosynthesis

Walport

Schofield

2018

The Chemical Record

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The 2-oxoglutarate (2OG) dependent oxygenases were first identified as having roles in the post-translational modification of procollagen in animals. Subsequently in plants and microbes, they were shown to have roles in the biosynthesis of many secondary metabolites, including signalling molecules and the penicillin/cephalosporin antibiotics. Crystallographic studies of microbial 2OG oxygenases and related enzymes, coupled to DNA sequence analyses, led to the prediction that 2OG oxygenases are widely distributed in aerobic biology. This personal account begins with examples of the roles of 2OG oxygenases in antibiotic biosynthesis, and then describes efforts to assign functions to other predicted 2OG oxygenases. In humans, 2OG oxygenases have been found to have roles in small molecule metabolism, as well as in the epigenetic regulation of protein and nucleic acid biosynthesis and function. The roles and functions of human 2OG oxygenases are compared, focussing on discussion of their substrate and product selectivities. The account aims to emphasize how scoping the substrate selectivity of, sometimes promiscuous, enzymes can provide insights into their functions and so enable therapeutic work.

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The Skp1 Protein from Toxoplasma Is Modified by a Cytoplasmic Prolyl 4-Hydroxylase Associated with Oxygen Sensing in the Social Amoeba Dictyostelium

Cited by 49 publications

References 43 publications

O -fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii

O -fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii

The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development

Adventures in Defining Roles of Oxygenases in the Regulation of Protein Biosynthesis

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