SMYD2 glutathionylation contributes to degradation of sarcomeric proteins

Reactive oxygen species (ROS) contribute to the etiology of multiple muscle-related diseases. There is emerging evidence that cellular stress can lead to destabilization of sarcomeres, the contractile unit of muscle. However, it is incompletely understood how cellular stress induces structural destabilization of sarcomeres. Here we report that glutathionylation of SMYD2 contributes to a loss of myofibril integrity and degradation of sarcomeric proteins mediated by MMP-2 and calpain 1. We used a clickable glutathione approach in a cardiomyocyte cell line and found selective glutathionylation of SMYD2 at Cys13. Biochemical analysis demonstrated that SMYD2 upon oxidation or glutathionylation at Cys13 loses its interaction with Hsp90 and N2A, a domain of titin. Upon dissociation from SMYD2, N2A or titin is degraded by activated MMP-2, suggesting a protective role of SMYD2 in sarcomere stability. Taken together, our results support that SMYD2 glutathionylation is a novel molecular mechanism by which ROS contribute to sarcomere destabilization.

of GS M4 with incubation of azido-Ala. Cells were lysed by freeze-thawing, and protein-free lysates were analyzed for thiol-concentrations by a bromobimane assay. Data represent the mean ± SD, n= 3 independent experiments. Difference is significant by two-tailed Student's unpaired ttest with Welch's correction (b) or one-way ANOVA followed by Tukey's post-hoc test (c), *p < 0.05 and N.S.= non-significant.
After incubation of azido-Ala, cells were subjected to H2O2 (a) or glucose deprivation (b-c).
Collected lysates were then subjected to click reaction with rhodamine-alkyne or biotin-alkyne. Glutathionylated proteins were analyzed by Western blotting with individual antibodies before and after pull-downs with streptavidin-agarose. (d) Analysis of SMYD2 oxidations with antibody that detects the sulfonic acid group. H9c2 cells expressing SMYD2 WT or C13S were treated with antimycin A (AMA), and the sulfonic acid formation in SMYD2 was probed by Western blotting after pull-down of SMYD2. (e) Validation of the sulfonic acid-antibody for detection of sulfonic acid.
To purified SMYD2 was added H2O2 (10 mM) without or with pre-treatment of iodoacetamide (I-AM) that blocks Cys residues. Blots are the representative of at least 3 independent experiments. Measurement of the level of SMYD2 glutathionylation. Purified SMYD2 WT or C13S was incubated with oxidized azido-glutathione (N3-GSSG-N3). At indicated time points, proteins were blocked with iodoacetamide. After click-reaction with 2-kD PEG-alkyne (c) or rhodamine-alkyne (d), proteins were precipitated to remove any remaining alkyne-derivatives and re-dissolved in PBS. Proteins in PBS were analyzed to measure rhodamine absorbance intensity at 550 nm to quantify the rhodamine concentration in comparison with a calibration curve. SMYD2 protein concentration was measured by gel analysis (ImageJ software to quantify the intensity of Coomassie bands) in comparison to the BSA calibration curve. Data represent the mean ± SD, n Difference is significant by one-way ANOVA followed by Tukey's post-hoc test (left panel) or twotailed Student's unpaired t-test with Welch's correction (right panel), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bars, 20 µm. or degradation of titin, titin was pull-downed from lysates with antibody that binds to a C-terminal region of titin (α-titin-CT), followed by Western blotting with antibody that binds to an N-terminal region of titin (α-titin-NT). Data represent the mean ± SD, n = 2 independent experiments.
Difference is significant by one-way ANOVA followed by Tukey's post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. with lysates (right). Data represent the mean ± SD, n = 2 independent experiments. Difference is significant by one-way ANOVA followed by Tukey's post-hoc test (a, d) or by two-tailed Student's unpaired t-test with Welch's correction (b-c), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (50 mM Tris-HCl, pH 8.0, and 1 M NaCl) to homogeneity and concentrated to 4-5 mg/ml. The purified protein was stored in a buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.2 mM βmercaptoethanol and 5% glycerol). The same approach was applied to express the GST-Hsp90 and GST-N2A where proteins were incubated with glutathione beads for purification, and proteins were stored in a buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.2 mM β-mercaptoethanol and 5% glycerol). GST on N2A domain was cleaved by HRV 3C Protease. Briefly, GST-N2A was incubated with the HRV 3C protease (enzyme/substrate = 1/50) overnight at 4°C in the supplied HRV 3C protease reaction buffer. The cleaved mixture was incubated with pre-washed glutathione beads for 2 h at 4°C. The cleaved N2A domain was then collected in the flow-through and stored in the same buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.2 mM β-mercaptoethanol and 5% glycerol). In vitro SMYD2 methyltransferase activity assay. SMYD2 methyltransferase activity was determined by measuring the amount of S-adenosylhomocysteine (SAH) generated from the enzymatic reaction. SAH was quantified by the multiple reactions monitoring (MRM) mode in LC-MS/MS with SAH-d4 as an internal standard. In MRM mode, the mass transition from the precursor to product was used to detect SAH (m/z 385 to 136) and SAH-d4 (m/z 389 to 136) with peak quantification. 5 The UV absorbance at 260 nm and a molar extinction coefficient 6 of 15,400 M -1 cm -1 were used to determine the concentartion of SAM and SAH. The calibration curve was made by plotting the integrated peak sizes versus SAH concentrations (10, 50, 100, 250 and 1000 nM). The activity assay was carried out at room temperature in a buffer (25 mM Tris-HCl, pH 8.0, and 0.01% Tween 20). SMYD2 (200 nM) was incubated with individual substrates, including p53 (25 μΜ), Hsp90 (4 μΜ), N2A (4 μΜ), Hsp90-GST (4 μΜ), and GST (4 μΜ), followed by addition of SAM (5 or 25 μΜ) at room temperature. Methylation was quenched at different time points by addition of TCA (5%).

Preparation of glutathionylated SMYD2
Detection of Hsp90 methylation. For detection of Hsp90 methylation, differentiated H9c2 cells without or with SMYD2 knockdown were treated with AMA (2 μg/mL). After lysis, Hsp90 was immunoprecipitated by using mouse Hsp90 antibody (BD transduction, Cat# 610418) and probed with rabbit mono-methyl lysine antibody (me-K) (Cell Signaling, Cat#14679) (1:1000). Similarly, purified Hsp90 or MMP-2 was incubated with SMYD2 and SAM at room temperature. Then reaction was quenched by addition of an SDS-loading buffer. Methylation of Hsp90 or MMP2 was probed by Western blotting with me-K antibody.