RhoA inactivation by S-nitrosylation regulates vascular smooth muscle contractive signaling

Area of Science:

• Biochemistry
• Cell Biology
• Physiology

Background:

• S-nitrosothiols (RSNOs) regulate cell signaling via thiol modification.
• The small G protein RhoA's GTP-binding domain contains critical cysteine residues.
• The role of RSNOs in modulating RhoA activity and downstream signaling remains unclear.

Purpose of the Study:

• To investigate if S-nitrosothiols modulate RhoA activity and downstream signals.
• To determine if specific cysteine residues in RhoA's GTP-binding domain are critical for regulation by RSNOs.

Main Methods:

• Used S-nitroso-L-cysteine (CSNO) to assess RhoA activation (GDP/GTP exchange, rhotekin binding, translocation).
• Generated and tested RhoA cysteine mutants (C20S, C26/20S, C16/20S) for resistance to CSNO.
• Measured MYPT1 and myosin light chain phosphorylation in smooth muscle cells and aortic tissue.

Main Results:

• CSNO inhibited RhoA activation, leading to RhoA nitrosylation and loss of activity.
• RhoA mutants C20S and C26/20S were resistant in vitro, but only C16/20S was resistant in intact cells.
• CSNO inhibited RhoA/Rho-kinase signaling, decreasing MYPT1 and myosin light chain phosphorylation independently of cGMP.
• Spermine NONOate's effects were cGMP-dependent, unlike CSNO's.

Conclusions:

• S-nitrosothiols inhibit RhoA/Rho-kinase signaling via modification of RhoA cysteine residues 16 and 20.
• This mechanism regulates myosin light chain phosphorylation and is cGMP-independent.
• Targeting RhoA cysteine modification by S-nitrosothiols could be a therapeutic strategy for vascular resistance disorders.