Using unnatural amino acid mutagenesis to probe the regulation of PRMT1

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Protein arginine methyltransferase 1 (PRMT1) is implicated in various diseases, including cancer and ALS.
• Regulatory mechanisms controlling PRMT1 activity are not well understood.
• Phosphorylation is a key post-translational modification that regulates protein function.

Purpose of the Study:

• To investigate the role of phosphorylation in regulating PRMT1 activity.
• To identify specific phosphorylation sites on PRMT1.
• To elucidate how phosphorylation affects PRMT1's substrate specificity and interactions.

Main Methods:

• Mass spectrometry to identify phosphorylated residues.
• Site-directed mutagenesis using natural and unnatural amino acids (e.g., pCmF, pBpF) to mimic phosphorylation.
• Cross-linking experiments with cell extracts to identify binding partners.
• In vitro assays to assess PRMT1 activity and binding.

Main Results:

• Tyrosine 291 (Tyr291), adjacent to the THW loop, was identified as a phosphorylation site.
• Phosphorylation at Tyr291 alters PRMT1's substrate specificity.
• Incorporation of phosphotyrosine mimics (pCmF, pBpF) confirmed the functional impact of Tyr291 phosphorylation.
• Cross-linking identified hnRNPA1 and hnRNP H3 as specific binding partners to the phosphorylated Tyr291 site.
• Tyr291 phosphorylation impairs PRMT1's ability to bind and methylate its protein substrates.

Conclusions:

• Tyr291 phosphorylation is a critical regulatory mechanism for PRMT1.
• This modification impacts both PRMT1's enzymatic activity and its protein-protein interaction profile.
• Understanding Tyr291 phosphorylation provides insights into PRMT1's role in disease pathogenesis and offers potential therapeutic targets.