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Identifying acetylated proteins in mitosis
1Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Abstract:
Histone deacetylase (HDAC) inhibitors are currently used in anticancer therapy to perturb genomic targets involved in gene transcriptional responses. However, the role of HDAC inhibitors on the acetylation of proteins outside of the transcriptional network has not been thoroughly assessed. We recently discovered that one of the HDACs, HDAC3, is localized on the mitotic spindle and regulates proper mitotic progression (1). To determine potential HDAC targets, we undertook a proteomics approach to search for acetylated proteins in mitosis (2). First, we synchronized cells in mitosis and used a polyclonal anti-acetyl-Lysine antiserum to immunoprecipitate acetylated proteins, followed by their identification by LC-ESI-MS/MS. We then confirmed the acetylation status of several mitotic proteins by anti-acetyl-Lysine immunoprecipitation with a monoclonal antibody followed by Western blot analyses of the proteins of interest. We further confirmed by a reciprocal immunoprecipitation with protein-specific antibody followed by Western blot analysis with another monoclonal anti-acetyl-Lysine antibody. Interestingly, the acetylation of a subset of the mitotic proteins can be further enhanced by treatment with apicidin, a small molecule inhibitor with specificity for HDAC3, suggesting that their acetylation may be regulated by HDAC3 in mitosis. In this chapter, we describe the various techniques using NudC as an example of an acetylated protein that is sensitive to apicidin treatment in mitosis.
Insights
Histone deacetylase inhibitors impact anticancer therapy. This study identifies novel mitotic protein acetylation targets regulated by HDAC3, enhancing our understanding of non-genomic roles in cell division.
Area of Science:
- Cell Biology
- Proteomics
- Molecular Biology
Background:
- Histone deacetylase (HDAC) inhibitors are utilized in cancer therapy targeting gene transcription.
- The role of HDACs in protein acetylation beyond genomic targets, particularly during mitosis, remains largely unexplored.
- HDAC3 has been identified as a key regulator of mitotic progression when localized on the mitotic spindle.
Purpose of the Study:
- To investigate potential non-histone protein targets of HDACs during mitosis.
- To identify acetylated proteins involved in mitosis using a proteomics approach.
- To explore the regulatory role of HDAC3 in mitotic protein acetylation.
Main Methods:
- Cell synchronization in mitosis followed by anti-acetyl-lysine immunoprecipitation.
- Identification of acetylated proteins using Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI-MS/MS).
- Validation of protein acetylation using Western blot analysis and reciprocal immunoprecipitation experiments.
Main Results:
- A subset of mitotic proteins was identified as acetylated.
- The acetylation status of several mitotic proteins was confirmed.
- Treatment with apicidin, an HDAC3 inhibitor, enhanced the acetylation of specific mitotic proteins, suggesting HDAC3 regulation.
Conclusions:
- HDAC3 plays a role in regulating the acetylation of non-histone proteins during mitosis.
- This study expands the known functions of HDACs to include the regulation of mitotic protein acetylation.
- NudC is presented as an example of an acetylated protein sensitive to apicidin treatment during mitosis.
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