Native detection of protein O-GlcNAcylation by gel electrophoresis

O-GlcNAcylation is an abundant and dynamic protein posttranslational modification (PTM), with crucial roles in metazoans. Studies of this modification are hampered by the lack of convenient methods for detecting native O-GlcNAcylation. Here, we describe a novel gel-based approach, Separation of O-GlcNAcylated Proteins by Polyacrylamide Gel Electrophoresis (SOPAGE), which enables detection of O-GlcNAc levels and dynamics

Acetazolamide-based fungal chitinase inhibitors

Chitin is an essential structural component of the fungal cell wall. Chitinases are thought to be important for fungal cell wall remodelling, and inhibition of these enzymes has been proposed as a potential strategy for development of novel anti-fungals. The fungal pathogen Aspergillus fumigatus possesses two distinct multi-gene chitinase families. Here we explore acetazolamide as a chemical scaffold for the inhibition of an A. fumigatus ‘plant-type’ chitinase. A co-crystal structure of AfChiA1 with acetazolamide was used to guide synthesis and screening of acetazolamide analogues that yielded SAR in agreement with these structural data. Although acetazolamide and its analogues are weak inhibitors of the enzyme, they have a high ligand efficiency and as such are interesting leads for future inhibitor development

Elevated O-GlcNAc levels activate epigenetically repressed genes and delay mouse ES cell differentiation without affecting naive to primed cell transition

The differentiation of mouse embryonic stem (ES) cells is controlled by the interaction of multiple signaling pathways, typically mediated by post-translational protein modifications. The addition of O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of nuclear and cytoplasmic proteins is one such modification (O-GlcNAcylation), whose function in ES cells is only now beginning to be elucidated. Here we demonstrate that the specific inhibition of O-GlcNAc hydrolase (Oga) causes increased levels of protein O-GlcNAcylation and impairs differentiation of mouse ES cells both in serum-free monolayer and in embryoid bodies (EBs). Use of reporter cell lines demonstrates that Oga inhibition leads to a reduction in the number of Sox1-expressing neural progenitors generated following induction of neural differentiation, as well as maintained expression of the ES cell marker Oct4 (Pou5f1). In EBs expression of mesodermal and endodermal markers is also delayed. However, the transition of naïve cells to primed pluripotency indicated by Rex1 (Zfp42), Nanog, Esrrb and Dppa3 downregulation and Fgf5 upregulation remains unchanged. Finally, we demonstrate that increased O-GlcNAcylation results in upregulation of genes normally epigenetically silenced in ES cells, supporting the emerging role for this protein modification in the regulation of histone modifications and DNA methylation. Stem Cells 2014

Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes

In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc),O-linked-GlcNAc transferase (OGT) catalyzes Ser/ThrO-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase-protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase-protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT