CHEMICAL SYNTHESIS OF SKP1 DERIVED GLYCOPEPTIDES FOR BIOCHEMICAL STUDIES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Eukaryotic organisms have evolved numerous mechanisms to sense O2 availability and regulate cellular function accordingly. A major pathway in animals is the O2-dependent prolyl 4-hydroxylation of the transcriptional factor subunit hypoxia inducible factor-1a (HIFa)1, which renders it a target of the E3VHLUb-ligase which signals subsequent degradation by the 26S-proteasome. In the mycetozoan Dictyostelium, the ortholog of animal HIFa-type prolyl 4-hydroxylases, P4H1, is also involved in O2 sensing. Genetic disruption of P4H1 increases the O2-requirement for culmination, the developmental process in which the multicellular slug converts to a fruiting body. In contrast, overexpression of P4H1 reduces the O2-requirement. These and other results suggest a model in which environmental O2 is a cue that influences the decision of the motile slug to differentiate, after migration to the soil surface, into a sessile fruiting body that promotes spore dispersal to potentially more favorable environments. Dictyostelium lacks an obvious ortholog of HIFa. A known target of Dictyostelium P4H1 is Skp1, an adaptor in E3SCFUb-ligases. SCF (Skp1/Cullin 1/F-box protein)- and VHL (von-Hippel Lindau)-type Ub-ligases are evolutionarily related and it is interesting that a subunit of the latter, VHL protein, recognizes a hydroxyproline target whereas in Dictyostelium, it is a subunit of the former type, Skp1, that is prolyl 4-hydroxylated. Subsequent to hydroxylation, HyPro143 is sequentially modified by 5 cytoplasmic glycosyltransferase activities, resulting in a glycan cap extending to up to 5 sugars. A previous study showed that the O2-requirement for a mutant disrupted in pgtA, required for addition of the second and subsequent sugars, was normal. This suggested that, except possibly for the first sugar, GlcNAc, the remainder of the glycosylation pathway fulfilled an alternative role.