N-terminal acetylation and methylation differentially affect the function of MYL9

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Post-translational modifications (PTMs) like acetylation and methylation can have opposing roles on the same residue.
• The interplay of PTMs on nonhistone proteins is not well understood.
• Myosin regulatory light chain 9 (MYL9) is the first identified protein with both N-terminal acetylation (Nα-acetyl) and N-terminal methylation (Nα-methyl) states in vivo.

Purpose of the Study:

• To investigate if Nα-acetylation and Nα-methylation of MYL9 create distinct proteoforms with different functions.
• To determine the regulatory roles of these Nα-PTMs on MYL9 activity.

Main Methods:

• Enzymatic assays to confirm MYL9 as a substrate for NRMT1 and NatA.
• Generation of MYL9 mutants specific for Nα-acetylation or Nα-methylation.
• Eukaryotic cell models to study the functional consequences of Nα-PTMs on MYL9.

Main Results:

• Neither Nα-acetylation nor Nα-methylation affects MYL9 protein stability.
• Exclusive Nα-acetylation promotes cytoplasmic roles, correlating with increased S19 phosphorylation.
• Exclusive Nα-methylation promotes nuclear roles as a transcription factor, enhancing DNA binding and reducing cytoskeletal interactions.

Conclusions:

• Nα-acetylation and Nα-methylation generate distinct MYL9 proteoforms with divergent cellular functions.
• These PTMs differentially regulate MYL9's localization, binding properties, and downstream activities.
• The study reveals a novel mechanism of proteoform diversification through competing N-terminal modifications.