Sensing and responding to changes in environmental O2 is critical for aerobic organisms. Metazoans accomplish this by destabilizing a transcription factor called hypoxia inducible factor (HIF) in O2 sufficiency. This is achieved by a O2-dependent prolyl 4-hydroxylase (PHD) that modifies proline residues in the HIF-1?ubunit making it the target of an E3 polyubiquitin ligase and ultimately dispatching it to the 26S-proteasome for degradation. PHDs are non-heme dioxygenases that use O2 and ketoglutarate to hydroxylate a proline residue and form succinate and CO2. Protozoans have PHDs but lack HIF and thus sense oxygen differently. In the social amoeba Dictyostelium, we discovered that its PHD modifies a proline in Skp1, which is a component of the SCF (Skp1/Cullin1/F-box protein/Rbx1) polyubiquitin ligase complex. Skp1 prolylhydroxylation does not affect its stability, but allows it to be modified by a series of glycosyltransferases. In turn glycosylation stabilizes the binding of Skp1 to F-box proteins essential for assembly of the SCF polyubiquitin ligases. Genome analysis and biochemical assays demonstrated that this Skp1 modification pathway is conserved in the protozoan parasite Toxoplasma gondii, but not in its human host. Moreover, loss of Toxoplasma PHD, or either of two glycosyltransferases, leads to a defect in parasite replication at low but physiological oxygen levels, and to changes in its Skp1 interactome. Because of its medical importance, we will focus on Toxoplasma and pursue three specific aims: i) Is Skp1 the key substrate for the Toxoplasma PHD and what is the contribution of novel glycosylation to PhyA activation? ii) How is Skp1 and polyubiquitin ligase assembly affected by prolyl hydroxylation? iii) How is the parasite PHD regulated and how can these data be used to develop parasite-specific pharmacological PHD inhibitors? To maximize progress in this renewal application, we have pooled the expertise of 3 independent investigators: i) a glycobiologist specialized in protozoan Skp1 and polyubiquitin ligases, ii) a Toxoplasma specialist experienced in hypoxia research, and iii) an organic chemist specializing in non-heme ?etoglutarate-dependent dioxygenases and their inhibition.

Public Health Relevance

The protozoan parasite Toxoplasma gondii latently infects 11-22% of Americans and the majority of some human groups. While the immune system of healthy individuals successfully holds this highly successful organism in a dormant state, the parasite is capable of reactivating causing toxoplasmosis when the immune system is compromised such as occurs during immune suppression therapy for transplantation or AIDS treatment. Infection of unexposed pregnant women is also devastating when parasites reach the fetus before her own immune system is activated. The lack of effective drug therapies makes toxoplasmosis exceedingly difficult to treat, underlying the importance of identifying novel drug targets that might eventually yield new treatments. The Skp1 modification pathway is one such candidate because of its multiple enzymes that contribute to parasite growth in cell culture models.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Intercellular Interactions Study Section (ICI)
Program Officer
Marino, Pamela
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Georgia
Schools of Arts and Sciences
United States
Zip Code
Gas-Pascual, Elisabet; Ichikawa, Hiroshi Travis; Sheikh, Mohammed Osman et al. (2018) CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology. J Biol Chem :
Saha, Sudeshna; Coleman, Bradley I; Dubey, Rashmi et al. (2017) Two Phosphoglucomutase Paralogs Facilitate Ionophore-Triggered Secretion of the Toxoplasma Micronemes. mSphere 2:
Rahman, Kazi; Mandalasi, Msano; Zhao, Peng et al. (2017) Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit. J Biol Chem 292:18644-18659
Rahman, Kazi; Zhao, Peng; Mandalasi, Msano et al. (2016) The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development. J Biol Chem 291:4268-80
West, Christopher M; Blader, Ira J (2015) Oxygen sensing by protozoans: how they catch their breath. Curr Opin Microbiol 26:41-7
Chinoy, Zoeisha S; Schafer, Christopher M; West, Christopher M et al. (2015) Chemical Synthesis of a Glycopeptide Derived from Skp1 for Probing Protein Specific Glycosylation. Chemistry 21:11779-87
Schafer, Christopher M; Sheikh, M Osman; Zhang, Dongmei et al. (2014) Novel regulation of Skp1 by the Dictyostelium AgtA ?-galactosyltransferase involves the Skp1-binding activity of its WD40 repeat domain. J Biol Chem 289:9076-88
Karunaratne, Chamini V; Weldeghiorghis, Thomas K; West, Christopher M et al. (2014) Conformational changes associated with post-translational modifications of Pro(143) in Skp1 of Dictyostelium--a dipeptide model system. J Am Chem Soc 136:15170-5
Feasley, Christa L; Hykollari, Alba; Paschinger, Katharina et al. (2013) N-glycomic and N-glycoproteomic studies in the social amoebae. Methods Mol Biol 983:205-29
Xu, Yuechi; Wang, Zhuo A; Green, Rebekah S et al. (2012) Role of the Skp1 prolyl-hydroxylation/glycosylation pathway in oxygen dependent submerged development of Dictyostelium. BMC Dev Biol 12:31

Showing the most recent 10 out of 18 publications