Acetylation of c-Myc at Lysine 148 Protects Neurons After Ischemia
Research Background
This study explores the role of the cancer-related transcription factor c-myc in penumbra neurons following ischemic stroke. Although the role of c-myc in cell death and survival has been recognized, research on its post-translational modifications, especially acetylation, in ischemic models remains insufficient. Investigating these modifications may have important clinical implications for controlling c-myc activity in the central nervous system. Current research on c-myc acetylation is mostly limited to non-neuronal cells. This study aims to investigate its expression during stroke recovery to explore the mechanisms regulated by acetylation.
Paper Source
The research findings were jointly completed by V.V. Guzenko, S.S. Bachurin, V.A. Dzreyan, A.M. Khaitin, Y.N. Kalyuzhnaya, and S.V. Demyanenko at Southern Federal University and Rostov State Medical University in Russia. The study was published in “Neuromolecular Medicine” Volume 26, Issue 8, 2024.
Research Process and Methods
Research Subjects and Experimental Methods
The experiments used CD-1 mice and adult male Wistar rats as models. Unilateral photoactivation of the right sensorimotor cortex was performed using a photothrombotic-induced cerebral ischemia model. Samples were taken at different time points (4 hours, 24 hours, 7 days) to analyze c-myc expression and its acetylation status.
Immunofluorescence Microscopy Techniques
Double immunofluorescence techniques were used to determine the expression and distribution of c-myc and its acetylated variants in penumbra cells after ischemia. Fluorescence intensity was calculated using image processing software, and protein co-localization was evaluated using the Jacop plugin. Western blotting and protein immunoprecipitation were used to confirm changes in c-myc expression and its acetylation levels in the nucleus and cytoplasm.
Molecular Dynamics Simulation
Molecular dynamics simulations were performed using the GROMACS software package to simulate the effects of c-myc protein acetylation at positions 148 and 323 on its conformation. The simulation results showed that acetylation at position 148 significantly affects the spatial structure of c-myc, potentially limiting its ability to pass through nuclear pores and leading to its accumulation in the cytoplasm.
Summary of Research Methods
The study used various inhibitors (such as the p300 acetyltransferase-specific inhibitor plumbagin and the deacetylase inhibitor mi192) to explore the effects of different enzymes on c-myc acetylation/deacetylation. The impact of these inhibitors on neuronal apoptosis was assessed using cell apoptosis detection (TUNEL).
Main Results
Changes in c-myc Expression in Penumbra Cells After Ischemia
In the acute phase after ischemia (4 hours and 24 hours), c-myc expression in the cerebral cortex nucleus increased significantly and was mainly acetylated at position 148 by p300 acetyltransferase. Subsequent immunofluorescence and Western blot results showed that the acetylation level of c-myc in neuronal cytoplasm increased significantly but did not exceed the control level during the recovery period.
Functional Significance of c-myc Acetylation at Position 148
Molecular dynamics simulations indicated that acetylation at position 148 leads to significant conformational changes in c-myc, potentially reducing its nuclear localization and thus decreasing its function as a transcription factor. Additionally, this acetylation may enhance c-myc’s interaction with unknown cytoplasmic proteins, promoting its accumulation in the cytoplasm, which may inhibit its pro-apoptotic effects.
Experimental Data Support
Experiments using p300 and sirt2 enzyme inhibitors found that the p300 inhibitor plumbagin and deacetylase inhibitors (such as the sirt2 inhibitor AK7) could affect the acetylation levels of c-myc in different subcellular structures, further supporting the importance of acetylation in regulating c-myc function.
Research Value and Significance
This study demonstrates that regulating c-myc acetylation at position 148 can significantly affect its function under ischemic injury conditions. Increasing c-myc acetylation levels by enhancing p300 acetyltransferase activity or using specific sirt2 inhibitors may become a potential therapeutic strategy to promote neuronal regeneration after ischemia. This research has important implications for neuroprotective therapy after ischemic brain injury and expands the understanding of cancer-associated protein functions in non-tumor environments.
Research Highlights
- Discovery of a Key Post-translational Modification Site: First revelation of the critical acetylation of c-myc at position 148 in cerebral ischemia.
- Innovative Methods: Combining immunoprecipitation, double immunofluorescence, and molecular dynamics simulations to systematically analyze the impact of c-myc acetylation on its function.
- Potential Clinical Applications: Using enzyme inhibitors to regulate c-myc acetylation provides a novel therapeutic strategy for ischemic brain injury.
This study not only deepens the understanding of c-myc’s mechanism of action in neurons but also offers new possibilities for future treatment of ischemic brain injury.