Related Experiment Videos
Peroxynitrite oxidation of tubulin sulfhydryls inhibits microtubule polymerization
Lisa M Landino1, Rifat Hasan, Ali McGaw
1Department of Chemistry, College of William & Mary, Williamsburg, Virginia 23187, USA. lmland@wm.edu
Abstract:
Considerable evidence both in vitro and in vivo implicates protein damage by peroxynitrite as a probable mechanism of cell death. Herein, we report that treatment of bovine brain microtubule protein, composed of tubulin and microtubule-associated proteins, with peroxynitrite led to a dose-dependent inhibition of microtubule polymerization. The extent of cysteine oxidation induced by peroxynitrite correlated well with inhibition of microtubule polymerization. Disulfide bonds between the subunits of the tubulin heterodimer were detected by Western blot as a result of peroxynitrite-induced cysteine oxidation. Addition of disulfide reducing agents including dithiothreitol and beta-mercaptoethanol restored a significant portion of the polymerization activity that was lost following peroxynitrite addition. Thus, peroxynitrite-induced disulfide bonds are at least partially responsible for the observed inhibition of polymerization. Sodium bicarbonate protected microtubule protein from the peroxynitrite-induced inhibition of polymerization. Tyrosine nitration of microtubule protein by 1 mM peroxynitrite increased approximately twofold when sodium bicarbonate was present whereas the extent of cysteine oxidation decreased from 7.5 to 6.3 mol cysteine/mol tubulin. These results indicate that cysteine oxidation of tubulin by peroxynitrite, rather than tyrosine nitration, is the primary mechanism of inhibition of microtubule polymerization.
Insights
Peroxynitrite damages microtubule proteins, inhibiting polymerization by forming disulfide bonds. Cysteine oxidation, not tyrosine nitration, is the main cause, but bicarbonate offers protection.
Area of Science:
- Biochemistry
- Cell Biology
- Neuroscience
Background:
- Protein damage by peroxynitrite is implicated in cell death.
- Microtubule polymerization is crucial for cellular structure and function.
Purpose of the Study:
- To investigate the mechanism by which peroxynitrite affects microtubule polymerization.
- To determine whether cysteine oxidation or tyrosine nitration is the primary cause of inhibition.
Main Methods:
- Treatment of bovine brain microtubule protein with varying concentrations of peroxynitrite.
- Assessing microtubule polymerization inhibition.
- Analyzing cysteine oxidation and tyrosine nitration using Western blot and quantifying modified residues.
- Testing the effect of disulfide reducing agents and sodium bicarbonate.
Main Results:
- Peroxynitrite caused dose-dependent inhibition of microtubule polymerization.
- Cysteine oxidation strongly correlated with polymerization inhibition.
- Peroxynitrite induced disulfide bond formation between tubulin subunits.
- Disulfide reducing agents partially restored polymerization activity.
- Sodium bicarbonate protected against polymerization inhibition, decreasing cysteine oxidation while increasing tyrosine nitration.
Conclusions:
- Peroxynitrite-induced cysteine oxidation, leading to disulfide bond formation, is the primary mechanism inhibiting microtubule polymerization.
- Tyrosine nitration is not the main driver of inhibition.
- Sodium bicarbonate offers a protective effect against peroxynitrite-induced microtubule damage.