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Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
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Evidence for small ubiquitin-like modifier-dependent nuclear import of the thymidylate biosynthesis pathway

Collynn F Woeller1, Donald D Anderson, Doletha M E Szebenyi

  • 1Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, USA.

The Journal of Biological Chemistry
|April 21, 2007
PubMed

Insights

Folate metabolism disruptions impact DNA and increase cancer risk. This study reveals how cytoplasmic serine hydroxymethyltransferase (cSHMT) SUMOylation in the nucleus directs folate to thymidylate synthesis, a process altered by the L474F polymorphism.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • Folate metabolism is crucial for cellular methylation and DNA synthesis, with disruptions linked to neural tube defects and cancer.
  • Genetic variations and folate deficiency can compromise one-carbon metabolism, affecting critical cellular processes.
  • Cytoplasmic serine hydroxymethyltransferase (cSHMT) plays a key role in folate metabolism, but its precise contribution to thymidylate biosynthesis is not fully understood.

Purpose of the Study:

  • To investigate the mechanism by which cSHMT-derived methylenetetrahydrofolate is preferentially directed to de novo thymidylate biosynthesis.
  • To explore the role of small ubiquitin-like modifier (SUMO)ylation in regulating cSHMT activity and localization.
  • To examine the impact of a common cSHMT genetic polymorphism (L474F) on enzyme function and cellular folate metabolism.

Main Methods:

  • In vitro assays to assess cSHMT interaction with UBC9 and its SUMOylation.
  • Detection of SUMOylated cSHMT in MCF-7 cell extracts during S phase.
  • Cellular localization studies of cSHMT during different phases of the cell cycle.
  • Analysis of the L474F-cSHMT polymorphism's effect on UBC9 interaction and SUMOylation.

Main Results:

  • cSHMT interacts with UBC9 and undergoes SUMOylation in vitro.
  • SUMOylated cSHMT is present in S phase cells and localizes to the nucleus and nuclear periphery during S and G(2)/M phases.
  • The L474F-cSHMT polymorphism impairs UBC9 interaction and inhibits cSHMT SUMOylation.
  • All three key enzymes in de novo thymidylate biosynthesis contain SUMOylation consensus sequences.

Conclusions:

  • Nuclear compartmentalization of the de novo thymidylate biosynthesis pathway explains the preferential use of cSHMT-derived folate units for thymidylate synthesis.
  • SUMOylation of cSHMT is a key regulatory mechanism influencing its nuclear localization and function in folate metabolism.
  • The L474F-cSHMT polymorphism may compromise nuclear folate metabolism efficiency, potentially increasing the risk for associated pathologies.

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