Glutathione transferase P1 is modified by palmitate

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Glutathione transferase P1 (GSTP1) is a key enzyme in cellular protection against electrophiles and regulates vital cellular processes.
• GSTP1 is typically cytosolic but can associate with the plasma membrane, suggesting compartment-specific functions.
• The role of post-translational modifications in regulating GSTP1 localization and function remains incompletely understood.

Purpose of the Study:

• To investigate the hypothesis that GSTP1 is palmitoylated.
• To determine if palmitoylation influences GSTP1's dynamic localization and cellular functions.
• To identify the sites and nature of GSTP1 palmitoylation.

Main Methods:

• Analysis of GSTP1 palmitoylation in MCF7 cells using cysteine-to-serine mutants.
• Treatment with sodium hydroxide (NaOH) to assess the stability of palmitate attachment.
• In vitro palmitoylation assays with purified GSTP1 and peptide sequencing.
• Subcellular fractionation and modified proximity ligation assays to determine localization.

Main Results:

• GSTP1 is palmitoylated in MCF7 cells, and this modification is retained in cysteine-mutant proteins.
• Palmitoylation of GSTP1 is resistant to NaOH treatment, indicating stable amide bond formation.
• Peptide sequencing identified S-palmitoylation at Cys48 and Cys102, and the rare N-palmitoylation at Lys103.
• Palmitoylated GSTP1 was detected in both membrane and cytosolic fractions, and associated with plasma membranes.

Conclusions:

• GSTP1 undergoes multi-site palmitoylation, including at non-cysteine residues (N-palmitoylation).
• Palmitoylation may play a role in regulating GSTP1's subcellular localization and diverse cellular functions.
• These findings reveal a novel regulatory mechanism for GSTP1 activity and localization.