Page 9 - T. Protein modification and regulation

Identification and Characterization of p300-mediated Acetylated Lysine
Residues in SERCA2a
Przemek A. Gorski , Kiyotake Ishikawa , Changwon Kho *
2
1,2
2
2
1 School of Korean Medicine, Pusan National University, Republic of Korea; Cardiovascular Research Center, Icahn School of Medicine
at Mount Sinai, USA, *Corresponding author; Presenter
BACKGROUND AIM
Impaired calcium uptake resulting from reduced expression and activity of Hypothesis: Increased acetylation of SERCA2a leads to
the cardiac sarco-endoplasmic reticulum calcium ATPase (SERCA2a) is
decreased calcium transport activity, resulting in alterations in
a hallmark of heart failure. The calcium sequestering activity of SERCA2a
contractility and global myocardial dysfunction.
is regulated by the phospholamban. However, other mechanisms of
SERCA2a regulation, including post-translational modifications (PTMs), Aim: To assess the role of lysine acetylation on SERCA2a function
have recently emerged. Our latest analysis of SERCA2a PTMs identified
in cardiomyocytes.
lysine acetylation, which might play an important role in regulating
SERCA2a activity. Indeed, we showed that SERCA2a is acetylated and
that this acetylation is more prominent in failing human hearts. Using a METHODS
cell-based screen, we found that p300 directly interacts with and • Mouse model: cardiac-specific Serca2 knock-out
acetylates SERCA2a. Also, we showed that p300 mediated acetylation
• Functional analysis: IonOptix calcium imaging
reduced SERCA2a ATPase activity. Analysis of acetylated SERCA2a by
• Biochemical analysis: Immunoprecipitation, Western blotting,
mass spectrometry revealed lysine residues in SERCA2a susceptible to ATPase activity assay, in vitro acetylation assay
acetylation by p300. Finally, our data demonstrate that p300 mediated
• Proteomic analysis: mass spectrometric analysis
acetylation of SERCA2a is a critical PTM that regulates calcium pump
• Protein structure-based computational prediction: PyMol
function, and it may provide invaluable information necessary to develop
new therapies for heart failure. • Statistics: Student’s t-test or ANOVA
RESULTS

I. p300 is increased in human heart failure II. p300 directly acetylates SERCA2a and inhibits its cellular function
A. Interaction C. Ca 2+ -dependent ATPase activity D. Contractility & Ca 2+ cycling of CMs

B. in vitro Acetylation

CTPB: p300 activator; L002: p300
Mean ± SD, * p < 0.05, NS: Not Significant Mean ± SD, * p < 0.05, NS: Not Significant inhibitor. Mean ± SD, NS: Not Significant
** p < 0.01; *** p < 0.001 vs DMSO;
## p < 0.01; ### p < 0.001 vs CTPB
III. Identification of p300-mediated acetylation sites in SERCA2a IV. Acetylation of K514 impairs cardiomyocyte function
Ca 2+ transient Ca 2+ reuptake

Maximal rate of contraction Maximal rate of relaxation

N=4 cardiomyocytes per group
Mean ± SD; NS: Not Significant; * p < 0.05; ** p < 0.01; *** p < 0.001
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
Our findings highlight the pathological Gorski PA et al. “Role of SIRT1 in modulating This work was supported by Pusan National
importance of post-translational acetylation of the sacro/endoplasmic reticulum University Research Grant, 2000.
2+
modifications and suggest that SERCA2a Ca -ATPase in heart failure” Circulation
activity may be manipulated by lysine Research. 2019;124(9):e63-e80. Contact information
acetylation.
Kho C et al. “SUMO1-dependent modulation of Changwon Kho, PhD., Assistant Professor
SERCA2a in heart failure” Nature. Email: khochangwon@pusan.ac.kr
2011;477:601-605. Tel: 82-51-510-8467