# Crosslinking-Assisted Substrate Identification for Lysine Demethylases

> **NIH NIH R01** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2021 · $34,400

## Abstract

Crosslinking-Assisted Substrate Identification for Lysine Demethylases
Abstract. Reversible lysine methylation on histone proteins constitutes a primary mechanism for gene
regulation. Of particular importance is the oxidative removal of the methyl groups by a conserved family of Fe2+-
and 2-ketoglutarate-dependent lysine demethylases (KDMs) that significantly affect transcriptional potential of a
gene. However, gene regulatory activity of KDMs is inherently complex due to their ability to demethylate a wide
range of non-histone proteins. This raises an important question: Are the biological functions of a given KDM
manifested through its histone or non-histone substrates or both? Currently, no method exists to characterize
KDM substrates in proteome-wide manner. To circumvent the challenge, we propose to develop a novel
chemoproteomic approach termed ‘crosslinking-assisted substrate identification (CASI)’. The CASI platform
involves engineering of a KDM active site with a photosensitive amino acid for light-mediated crosslinking with
the bound substrates followed by identification of the crosslinked species using quantitative mass spectrometry.
Using development- and cancer-relevant lysine demethylase 4A (KDM4A) as paradigm, we show that such
engineering approach is feasible. We plan to extend this approach to all the members of the KDM4 family and
to identify their distinct substrates from human cells. Subsequent biochemical studies of the newly identified non-
histone substrates would lead to improved understanding of how KDM4-mediated demethylation of critical
cellular proteins controls protein-protein interactions, reprograms gene expression, repairs DNA damage and
promotes tumor metastasis. We anticipate the CASI approach to be highly general and applicable to any
chromatin-modifying enzyme to elucidate their functions in a manner not attainable by existing methods.

## Key facts

- **NIH application ID:** 10388729
- **Project number:** 3R01GM130752-02S1
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Kabirul Islam
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $34,400
- **Award type:** 3
- **Project period:** 2020-08-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10388729

## Citation

> US National Institutes of Health, RePORTER application 10388729, Crosslinking-Assisted Substrate Identification for Lysine Demethylases (3R01GM130752-02S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10388729. Licensed CC0.

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