Cell differentiation depends fundamentally on the turnover of selected regulatory and structural proteins. This universal aspect of development is mediated in large part by polyubiquitination, which targets proteins for disposal in the 26S-proteasome. A major group of polyubiquitin ligases, the SCF (Skp1-cullin1-Fbox) subclass of cullin-RING-type E3 ubiquitin (Ub)-ligases (CRLs), has been directly implicated in numerous physiological (including cell cycle) and developmental processes. A critical feature is their use o a specificity factor for selecting targets, following a priming event such as phosphorylation. Considerable evidence indicates that CRL Ub-ligases are also regulated. Our findings in the model organism Dictyostelium reveal new and novel mechanisms for regulating the SCF class itself. These mechanisms appear to be widespread in protists including many important human pathogens. The novel mechanisms involve covalent modification of the Skp1 adaptor by prolylhydroxylation and subsequent serial modification by 5 sugars to ultimately form a pentasaccharide. We defined the genetics and enzymology of the pathway in the last and earlier project periods. These studies also revealed striking changes in the O2 dependence of development which we traced back to an O2 sensor function of the prolyl 4-hydroxylase analogous to a corresponding process in humans. We also discovered that successive glycosylation steps modulate O2 sensing, and that the final glycosyltransferase in the pathway, AgtA, has enzyme-independent functions that are also necessary for proper development. Our newest findings now suggest that these modifications alter the conformation of Skp1, which inhibits its homodimerization and promotes binding of Skp1 to Fbox proteins, leading to their auto-polyubiquitination and premature degradation. Furthermore, AgtA competitively binds unmodified Skp1, by a novel self-limiting mechanism mediated by glycosylation. Because these interactions pertain to the assembly of E3SCFUb-ligases, we hypothesize that the Skp1 modification enzymes ultimately control the ubiquitination of many of the ~50 predicted Fbox proteins, most of which are differentially regulated during development, and potentially their target substrates as well. The goal of this project is to define the biochemical mechanism of how this occurs. This model is important be- cause it offers a novel mode of specific regulation of E3SCFUb-ligases with major impact on development, and the occurrence of the 6-enzyme pathway in pathogenic protists presents a large drug target for future exploitation. The studies will be conducted in Dictyostelium, an experimentally facile organism where we have developed invaluable tools to test the hypothesis.
Aim 1 will investigate how hydroxylation, glycosylation, and AgtA affect assembly of SCF complexes and their E3 Ub-ligase activities in vitro.
Aim 2 will investigate the relevance of the findings to Skp1 complex assembly and activities in cells using immunoprecipitation and microscopy.
Aim 3 will employ gene and inhibitor synthetic studies to address the linkage of Skp1 modification to ubiquitination and degradation activities in developmental regulation.

Public Health Relevance

The greatest potential biomedical relevance for the novel regulation of E3SCFUb-ligases is to the protozoan pathogens that possess the pathway genes, including plant pathogens of the Phytophthora group, which impact on human nutrition (e.g., soybean production), Acanthamoeba, which causes amoebic keratitis and encephalitis, and apicomplexans that include the agent for toxoplasmosis (Toxoplasma gondii), an important disease of humans and livestock. In addition, emerging evidence suggests that a vector of human Legionnaires disease, Legionella pneumophila, depends on Skp1 for proliferation in Dictyostelium, a potential reservoir, and other hosts. We anticipate that our insights from studies in the model organism Dictyostelium will inspire novel tests of prolyl 4-hydroxylase inhibitors tha are emerging from studies of the animal enzyme, toward the control of infectious disease.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM037539-22
Application #
8697057
Study Section
Intercellular Interactions Study Section (ICI)
Program Officer
Marino, Pamela
Project Start
Project End
Budget Start
Budget End
Support Year
22
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Oklahoma Health Sciences Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Oklahoma City
State
OK
Country
United States
Zip Code
73117
Sheikh, M Osman; Xu, Yuechi; van der Wel, Hanke et al. (2015) Glycosylation of Skp1 promotes formation of Skp1-cullin-1-F-box protein complexes in dictyostelium. Mol Cell Proteomics 14:66-80
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
Sheikh, M Osman; Schafer, Christopher M; Powell, John T et al. (2014) Glycosylation of Skp1 affects its conformation and promotes binding to a model f-box protein. Biochemistry 53:1657-69
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
Zhang, Dongmei; van der Wel, Hanke; Johnson, Jennifer M et al. (2012) Skp1 prolyl 4-hydroxylase of dictyostelium mediates glycosylation-independent and -dependent responses to O2 without affecting Skp1 stability. J Biol Chem 287:2006-16
van der Wel, Hanke; Johnson, Jennifer M; Xu, Yuechi et al. (2011) Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and ?-N-acetylglucosaminyltransferase enzymes involved in O? signaling in dictyostelium. Biochemistry 50:1700-13
Wang, Zhuo A; Singh, Divyendu; van der Wel, Hanke et al. (2011) Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium. Dev Biol 349:283-95
West, Christopher M; Wang, Zhuo A; van der Wel, Hanke (2010) A cytoplasmic prolyl hydroxylation and glycosylation pathway modifies Skp1 and regulates O2-dependent development in Dictyostelium. Biochim Biophys Acta 1800:160-71
Olszewski, Neil E; West, Christopher M; Sassi, Slim O et al. (2010) O-GlcNAc protein modification in plants: Evolution and function. Biochim Biophys Acta 1800:49-56
Qian, Yi; West, Christopher M; Kornfeld, Stuart (2010) UDP-GlcNAc:Glycoprotein N-acetylglucosamine-1-phosphotransferase mediates the initial step in the formation of the methylphosphomannosyl residues on the high mannose oligosaccharides of Dictyostelium discoideum glycoproteins. Biochem Biophys Res Commun 393:678-81

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