Oxygen sensing by protozoans: how they catch their breath

West, Christopher M.; Blader, Ira J.

doi:10.1016/j.mib.2015.04.006

Cited by 34 publications

(59 citation statements)

References 47 publications

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“…The results with singly substituted CODD peptides and the hybrid peptides (with both wild‐type tPHD2 and the R396A variant) support the proposal that the interactions between the C terminus of the ODDs and the C‐terminal region of tPHD2, including α4, are important in enhancing CODD relative to NODD hydroxylation; as observed here and previously, CODD is a better substrate than NODD. The reduced reactivity of R396A tPHD2 with CODD, but not NODD, is consistent with the observed salt bridge between R396 tPHD2 (part of the α4 helix) and D571 CODD (Figure S3 B).…”

Section: Resultssupporting

confidence: 88%

Studies on the Substrate Selectivity of the Hypoxia‐Inducible Factor Prolyl Hydroxylase 2 Catalytic Domain

et al. 2018

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In animals, the response to chronic hypoxia is mediated by upregulation of the α,β-heterodimeric hypoxia-inducible factors (HIFs). Levels of HIFα isoforms, but not HIFβ, are regulated by their post-translational modification as catalysed by prolyl hydroxylase domain enzymes (PHDs). Different roles for the human HIF-1/2α isoforms and their two oxygen-dependent degradation domains (ODDs) are proposed. We report kinetic and NMR analyses of the ODD selectivity of the catalytic domain of wild-type PHD2 (which is conserved in nearly all animals) and clinically observed variants. Studies using Ala scanning and "hybrid" ODD peptides imply that the relatively rigid conformation of the (hydroxylated) proline plays an important role in ODD binding. They also reveal differential roles in binding for the residues on the N- and C-terminal sides of the substrate proline. The overall results indicate how the PHDs achieve selectivity for HIFα ODDs and might be of use in identifying substrate-selective PHD inhibitors.

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Section: Resultssupporting

confidence: 88%

Studies on the Substrate Selectivity of the Hypoxia‐Inducible Factor Prolyl Hydroxylase 2 Catalytic Domain

et al. 2018

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“…Given the large variety of substrates known to be recognized by yeast, plant, and human F-box proteins, further studies are needed to evaluate whether other processes required for plaque formation, such as binding of the parasite to host cells, ingress and/or egress are affected. In future studies, it will be interesting to evaluate whether any of these potential mechanisms are more severely affected in low O 2 or altered metabolic states that are anticipated to influence Skp1 modification enzyme activities in cells (11).…”

Section: Discussionmentioning

confidence: 99%

“…In Dictyostelium, hydroxylation alone partially rescues O 2 -dependent development (10). Full recovery depends, however, on full glycosylation (11), and glycosylation is required to promote efficient assembly of the Skp1/F-box protein heterodimer, based on interactome studies (12). Therefore, we sought direct evidence for Hyp-dependent glycosylation of Toxoplasma Skp1 and the role of the predicted glycosyltransferases, and we used disruption strains of the predicted glycosyltransferase genes to test their contribution to parasite proliferation.…”

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confidence: 99%

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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“…A novel glycosylation pathway whose glycosyltransferases (GTs) reside in the cytoplasm evolved early in eukaryotic evolution and persisted in most branches of protist radiation before disappearing in advanced fungi, land plants, and animals. Studies of the pathway in the amoebozoan Dictyostelium discoideum and the apicomplexan Toxoplasma gondii show a role in O 2 -sensing, owing to the requirement for an O 2dependent prolyl 4-hydroxylase, PhyA, that modifies a critical prolyl residue of Skp1, a subunit of the SCF class of E3 ubiquitin (Ub) ligases (West and Blader, 2015). PhyA action generates a 4(trans)hydroxyproline (Hyp) residue, which serves as the attachment point of the pentasaccharide that is assembled by the sequential action of five GT activities.…”

Section: Introductionmentioning

confidence: 99%

“…Thus Skp1 is a rare example of a glycoprotein that is not only glycosylated in a complex manner in the cytoplasm, but also remains to function in this compartment and the nucleus, rather than being exported (West and Hart, 2017). Skp1 is the only protein that is detectably glycosylated by any of the five GT activities in D. discoideum (West and Blader, 2015), which is at a variance from most GTs, which typically modify a range of proteins. Genetic manipulation of Skp1 expression, substitution of the target Pro, and genetic interactions with a Skp1 GT, affect D. discoideum O 2 -sensing in ways that are consistent with Skp1 being the functional target of prolyl hydroxylation in O 2 -sensing (Wang et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

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

Mandalasi¹,

Kim²,

Thieker³

et al. 2019

Preprint

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Skp1, a subunit of E3-Skp1/Cullin-1/F-box protein ubiquitin ligases, is uniquely modified in protists by an O 2 -dependent prolyl hydroxylase, which forms the attachment site for a novel pentasaccharide. Mutational studies demonstrate the importance of the core glycan for growth of the parasite Toxoplasma gondii in fibroblasts, but the significance of the non-reducing terminal sugar is unknown. Here we investigated a homolog of glycogenin, an enzyme that can initiate and prime glycogen synthesis in yeast and animals. Gat1 is required for pentasaccharide assembly in cells and catalyzes the addition of an α galactose in 3-linkage to the subterminal α 3-linked glucose residue in vitro. A strong selectivity of Gat1 for Skp1 in extracts is consistent with evidence that Skp1 is the sole target of the glycosyltransferase pathway. gat1-disruption resulted in slow growth indicating the importance of the complete glycan. Molecular dynamics simulations suggested that the full glycan helps organize Skp1 as previously described in the amoebozoan Dictyostelium where a distinct glycosyltransferase assembles a different terminal disaccharide. The crystal structure of Gat1 from the plant pathogen Pythium ultimum confirmed the striking similarity to glycogenin, with differences in the active sites providing an explanation for its distinct substrate preference and regiospecificity. Gat1 also exhibited low α -glucosyltransferase activity like glycogenin, but autoglycosylation was not detected and gat1-disruption revealed no effect on starch accumulation in Toxoplasma. A phylogenetic analysis suggested that Gat1 was a progenitor of glycogenin, and acquired its role in glycogen formation following the ancestral disappearance of the underlying Skp1 glycosyltransferase Glt1 prior to amoebozoan evolution.

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Oxygen sensing by protozoans: how they catch their breath

Cited by 34 publications

References 47 publications

Studies on the Substrate Selectivity of the Hypoxia‐Inducible Factor Prolyl Hydroxylase 2 Catalytic Domain

Studies on the Substrate Selectivity of the Hypoxia‐Inducible Factor Prolyl Hydroxylase 2 Catalytic Domain

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

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

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