3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• 3-Bromopyruvate (3BrPA) is an antitumor agent targeting thiol groups of enzymes.
• Its proposed use in cancer treatment stems from selective reactivity with tumor cell metabolic enzymes.

Purpose of the Study:

• To investigate 3-Bromopyruvate (3BrPA) as a potential inhibitor of sarco/endoplasmic reticulum calcium (Ca2+) ATPase (SERCA) type 1.
• To elucidate the mechanism of 3BrPA interaction with SERCA activity.

Main Methods:

• Incubation of sarco/endoplasmic reticulum vesicles (SRV) with varying concentrations of 3BrPA.
• Assay of SERCA ATPase activity and Ca2+ uptake in the presence and absence of 3BrPA and reduced glutathione (GSH).
• Analysis of cysteine residue modification.

Main Results:

• 150 μM 3BrPA significantly inhibited SERCA Ca2+ uptake by 80%, while ATPase activity remained unaffected.
• 3BrPA uncouples ATP hydrolysis from Ca2+ transport in SERCA.
• 2 mM GSH increased SERCA ATPase activity by 40% and protected Ca2+ uptake from 3BrPA inhibition.
• GSH and 3BrPA treatment led to decreased reduced cysteine levels, indicating pyruvylation or S-glutathiolation at critical SERCA cysteine residues.

Conclusions:

• 3-Bromopyruvate (3BrPA) selectively inhibits SERCA Ca2+ transport, uncoupling it from ATP hydrolysis.
• Reduced glutathione (GSH) mitigates 3BrPA's inhibitory effects on Ca2+ uptake.
• The findings suggest that 3BrPA targets critical cysteine residues in SERCA, potentially through pyruvylation or S-glutathiolation, impacting its function.