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Understanding the mechanism of adaptor protein engagement by OGT and its functional effects on glycosylation

Award Number: R15GM147888
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/01/2022
PERIOD OF PERFORMANCE END DATE: 02/28/2029

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Understanding the mechanism of adaptor protein engagement by OGT and its functional effects on glycosylation - PROJECT SUMMARY/ABSTRACT The identification of O-linked beta-N-acetylglucosamine (O-GlcNAc) modified proteins in the nucleus and cytoplasm overturned the paradigm that glycosylated proteins are only found in the secretory pathway. Since then, O-GlcNAc modifications, installed by the O-GlcNAc transferase (OGT) enzyme, have been identified on proteins involved in almost every cellular process. O-GlcNAc levels rise upon increase of glucose levels, and perturbations in protein O-GlcNAcylation has been implicated in diseases caused by dysregulation of proteostasis, such as cancer and neurodegeneration. It has been proposed that methods to regulate O- GlcNAcylation levels on targeted substrates would be therapeutically valuable. To date, over four thousand protein targets have been identified, however the mechanisms by which OGT chooses those substrates eludes scientists, hindering the development of selective therapeutic interventions. Substrate selection does not occur at the active site of OGT. Instead, OGT’s N-terminal tetratricopeptide repeat (TPR) domain governs substrate selection through two proposed mechanisms, either through 1) intrinsic interactions with substrates and/or 2) interactions with intermediary proteins, called adaptors, that alter OGT’s enzymatic activity towards subsets of substrates. The TPR domain contains 13.5 repeats that form a unique superhelix with two 100 Å long binding surfaces, the concave, lumenal surface that engages in direct substrate binding and a convex, solvent-exposed surface that we hypothesize interacts with non-substrate proteins. While much is known about intrinsic substrate binding, adaptor-mediated substrate selection mechanisms are poorly understood due to the limited tools for selectively capturing non-substrate interactions. We have developed a photoactivatable OGT library in which UV-active unnatural amino acids have been incorporated along the solvent-exposed surface of the TPR domain enabling the differentiation of the two binding surfaces. This library has identified the binding sites for a known OGT adaptor, CARM1, and covalently captured protein interactors along TPR 1 involved in RNA processing. I propose experiments to determine the atomic level details by which TPR-specific adaptors modulate OGT’s activity towards specific substrates involved in post-transcriptional regulation. In Aim 1, we will use biochemical and structural techniques to determine the composition of two distinct OGT-CARM1 complexes and mutagenesis strategies and glycosylation profiling to interrogate the functional role of each complex on OGT’s substrate selectivity. In Aim 2, we will use photocrosslinking assays and in cellulo glycosylation profiling to determine whether RNA-binding proteins identified in a recent proteomics screen in our lab are OGT adaptors and how their interactions with TPR 1 drive targeted OGT glycosylation within the post-transcriptional landscape. Results from this study will identify how OGT’s activity towards RNA-binding proteins is modulated by adaptor interactions at different sites along the TPR domain and can be manipulated by disrupting these interactions. This information will enable the advancement of strategies to selectively target OGT’s activity in post-transcriptional regulation.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $315,554 )
2026	2026	ST. OLAF COLLEGE	1520 SAINT OLAF AVE	NORTHFIELD	MN	55057	RICE	USA	Biomedical Research and Research Training	002	2	3/6/2026	COMPETING CONTINUATION	$0
2026	2026	ST. OLAF COLLEGE	1520 SAINT OLAF AVE	NORTHFIELD	MN	55057	RICE	USA	Biomedical Research and Research Training	001	2	3/1/2026	COMPETING CONTINUATION	$315,554
2026	2022	ST. OLAF COLLEGE	1520 SAINT OLAF AVE	NORTHFIELD	MN	55057	RICE	USA	Biomedical Research and Research Training	000	1	2/9/2026	NEW	$0
														Subtotal = $315,554

Issue Date FY: 2023 ( Subtotal = $64,637 )
2023	2023	ST. OLAF COLLEGE	1520 SAINT OLAF AVE	NORTHFIELD	MN	55057	RICE	USA	Biomedical Research and Research Training	001	1	6/22/2023	SUPPLEMENT FOR EXPANSION	$64,637
2023	2022	ST. OLAF COLLEGE	1520 SAINT OLAF AVE	NORTHFIELD	MN	55057	RICE	USA	Biomedical Research and Research Training	000	1	11/8/2022	NEW	$0
														Subtotal = $64,637

Issue Date FY: 2022 ( Subtotal = $321,331 )
2022	2022	ST. OLAF COLLEGE CORP	1520 ST OLAF AVE	NORTHFIELD	MN	55057	RICE	USA	Biomedical Research and Research Training	000	1	8/12/2022	NEW	$321,331
														Subtotal = $321,331

Grand Total All Awards = $701,522

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Understanding the mechanism of adaptor protein engagement by OGT and its functional effects on glycosylation

Award Number: R15GM147888

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 09/01/2022

PERIOD OF PERFORMANCE END DATE: 02/28/2029

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