CTPS2 regulates CTP synthetase activity by interacting with CTPS1.

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Cytidine triphosphate synthetases (CTPS) 1 and 2 are crucial for de novo CTP nucleotide production, essential for DNA replication and cell proliferation.
• CTPS1 and CTPS2 are co-expressed, share structural homology, and form enzymatically active homotetramers that aggregate into cytoophidia.
• The role of cytoophidia in cell proliferation and the interaction between CTPS1 and CTPS2 remain incompletely understood.

Purpose of the Study:

• To investigate the co-localization, interaction, and functional relationship between CTPS1 and CTPS2.
• To determine the necessity of cytoophidia formation for cell proliferation.
• To elucidate the impact of CTPS2 on CTPS1 enzymatic activity and feedback regulation.

Main Methods:

• Co-expression of CTPS1 and CTPS2 to observe co-localization within cytoophidia.
• Expression of catalytically inactive CTPS1 and CTPS2 mutants (CTPS1^H355A, CTPS2^H355A) to assess cytoophidia necessity for proliferation.
• Co-immunoprecipitation assays to detect direct interaction between CTPS1 and CTPS2.
• Enzyme activity assays to measure CTPS1 activity in the presence of CTPS2 and CTP/product feedback inhibition.

Main Results:

• CTPS1 and CTPS2 co-localized in cytoophidia, with CTPS2 filament formation dependent on CTPS1 expression.
• Cytoophidia formation is not essential for cell proliferation, as demonstrated by the sustained proliferation of CTPS1^H355A and CTPS2^H355A mutants.
• CTPS1 and CTPS2 directly interact independent of polymerization and cytoophidia formation.
• CTPS2 association decreases CTPS1 enzymatic activity and increases its sensitivity to CTP/product feedback inhibition, indicating modulation of CTPS activity by CTPS2.

Conclusions:

• CTPS1 and CTPS2 interact directly, forming complexes that modulate enzymatic activity and feedback regulation, independent of cytoophidia.
• Cytoophidia are dispensable for cell proliferation, suggesting alternative mechanisms for CTP synthesis regulation.
• The findings suggest that CTPS1 and CTPS2 can directly regulate each other through heterotetramerization, revealing a novel layer of CTP metabolism control.