Dataset Information

Targeted deglycosylation with a nanobody-directed split OGA in living cells

ABSTRACT: O-GlcNAc is an essential and dynamic post-translational modification present on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a target protein in cells is critical yet challenging without disturbing O-GlcNAc globally or after extensive glycosite mapping. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as a targeted O-GlcNAc eraser for protein-selective O-GlcNAc removal in cells. Through systematic screening, we identified the essential domains of OGA and afforded a split OGA with limited activity, which selectively reinstated deglycosylation activity on the target protein upon fusion of a nanobody in living cells. We demonstrate the generality of the O-GlcNAc eraser against a series of target proteins and reveal that O-GlcNAc stabilizes the transcription factor c-Jun and promotes its interaction with c-Fos. Thus, nanobody-directed split OGA speeds the selective removal and functional evaluation of O-GlcNAc on individual proteins via an approach that is generalizable to a broader array of post-translational modifications.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, Kidney

SUBMITTER: Christina Woo

LAB HEAD: Christina Woo

PROVIDER: PXD018914 | Pride | 2022-02-15

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	190930L_SAM6100_YG147_TF_TMT_final no normal.pride.mgf.gz	Mgf
	190930L_SAM6100_YG147_TF_TMT_finalnonormal.mzML	Mzml
	Yun_OGA_mzid.mzid.gz	Mzid
	Yun_OGA_mzid.pride.mztab.gz	Mztab
	Yun_OGA_rawdata.raw	Raw

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Publications

Developing and Testing of an Oral Health Screening Tool for Midwives to Assess Pregnant Woman.

George Ajesh A Ajwani Shilpi S Johnson Maree M Dahlen Hannah H Blinkhorn Anthony A Bhole Sameer S Ellis Sharon S Zheng Catherine C Dawes William W

Health care for women international 20141211 10

Maternal oral health is important, and midwives are encouraged to screen women for dental problems. We aimed to develop and test a midwifery oral health screening tool. A three-item tool was tested as part of a trial involving 300 women in Southwestern Sydney. A two-item combination showed better sensitivity (98%) and had a positive predictive value of 88%. Specificity was 40%, and negative predictive value was 80%. A two-item screening tool has been identified that is sensitive to identifying d ...[more]

PMID: 25299615

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Project description:Drosophila development is a complex and dynamic process regulated, in part, by members of the Polycomb (Pc), Trithorax (Trx) and Compass chromatin modifier complexes. O-GlcNAc Transferase (OGT/SXC) is essential for Pc repression suggesting that the O-GlcNAcylation of proteins plays a key role in regulating development. OGT transfers N-acetyl-D-glucosamine (GlcNAc) onto hydroxyl groups of serine or threonine residues of key transcriptional regulators using the nutrient-derived UDP-GlcNAc as a substrate, which is dynamically removed by O-GlcNAcase (OGA). We performed ChIP-chip and microarray analysis after OGT or OGA RNAi knockdown in Drosophila S2 cells and found that O-GlcNAc was elevated genome wide particularly at genes related to mitosis and cell cycle in OGA RNAi cells, but not at sites co-occupied by Pc member Pleiohomeotic (Pho), such as the Hox and NK homeobox gene clusters. Microarray analysis suggested that altered O-GlcNAc cycling perturbed the expression of genes associated with morphogenesis and cell cycle regulation. To examine the in vivo consequences of disturbed O-GlcNAc cycling in the whole animal, we produced a null allele of oga (ogadel.1) in Drosophila. Epigenetic activators including Trx group members Trithorax (Trx), Absent small or homeotic discs 1 (Ash1) and Compass member Set1 histone methyltransferases are O-GlcNAc modified in ogadel.1 mutants. ogadel.1 mutants displayed altered expression of a distinct set of cell cycle related genes in ovaries. Our results suggest that the loss of OGA could affect epigenetic machinery by accumulating O-GlcNAc on numerous chromatin factors including Trx, Ash1 and Set1 in Drosophila. We performed affymetrix tilingarray analysis after OGT or OGA RNAi knockdown in Drosophila S2 cells to find if that O-GlcNAc was elevated genome wide particularly at genes related to mitosis and cell cycle in OGA RNAi cells, but not at sites co-occupied by Pc member Pleiohomeotic (Pho), ------------------------------- This represents the gene expression component only