# Chemical Methods for Dissecting Protein Glutathionylation in Sarcomere

> **NIH NIH R01** · WAYNE STATE UNIVERSITY · 2021 · $363,880

## Abstract

Summary/Abstract
This application describes chemical approaches for determining the role of protein cysteine glutathionylation in
the sarcomere. Sarcomere is a basic unit of myofibrils in muscle. Sarcomere contains numerous sarcomeric
proteins, including titin, actin and myosin, that form a highly organized structure for continuous contraction of
muscle. In muscle cell, the reactive oxygen species (ROS) are emerging as critical signaling molecules that
strongly contribute to physiology and pathology associated with heart function. However, the precise molecular
target proteins of ROS and their redox-based regulatory mechanisms that affect sarcomere stability and integrity
remain unknown. Glutathionylation is one of the major protein cysteine oxidative modifications that mediate the
role of ROS in redox signaling and oxidative stress. This application is based on our recently developed chemical
approach, i.e. clickable glutathione for identification and characterization of glutathionylation. SET and MYND
domain-containing protein 2 (SMYD2) is an abundant protein in heart and skeletal muscle. With clickable
glutathione, we found that SMYD2 is selectively glutathionylated at C13, and SMYD2 C13 glutathionylation is a
crucial mechanism by which ROS induce sarcomere destabilization in cardiomyocytes. The main goal of
application is to identify sarcomeric proteins, including SMYD2, that are susceptible to glutathionylation in
response to ROS and to characterize functional roles of protein glutathionylation in regulating sarcomere stability.
There are three specific aims. First, we plan to couple clickable glutathione with mass analysis to identify
glutathionylation of SMYD2 and other sarcomeric proteins in response to ischemic conditions. Clickable
glutathione approach will be used in H9c2 cell line with isotopic-labelled azido-Ala and cleavable biotin-alkyne
for quantitative mass analysis of glutathionylated proteins under oxygen-glucose-deprivation. Second, we will
determine sarcomere stability and integrity resulting from SMYD2 C13 glutathionylation. We will determine
sarcomere stability in myocytes in response to ROS by fluorescence imaging of sarcomeric proteins, including
myosin and actin. Also, we will couple clickable glutathione with proximity ligation for visualizing localization of
glutathionylated SMYD2. Third, we plan to determine the molecular mechanism by which SMYD2
glutathionylation leads to sarcomere destabilization. We will synthesize site-specifically glutathionylated SMYD2,
which will be used for characterizing structural and functional changes of SMYD2 C13 glutathionylation with
subsequent cellular studies. Taken together, these studies will uncover the key molecular target protein and
molecular mechanisms by which ROS contribute to muscle dysfunction.

## Key facts

- **NIH application ID:** 10171883
- **Project number:** 5R01HL131740-05
- **Recipient organization:** WAYNE STATE UNIVERSITY
- **Principal Investigator:** Young-Hoon Ahn
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $363,880
- **Award type:** 5
- **Project period:** 2017-08-15 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10171883

## Citation

> US National Institutes of Health, RePORTER application 10171883, Chemical Methods for Dissecting Protein Glutathionylation in Sarcomere (5R01HL131740-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10171883. Licensed CC0.

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