Uterine smooth muscle S-nitrosylproteome in pregnancy

Abstract

The molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor are not completely known. Nitric oxide relaxes uterine smooth muscle in a manner disparate from that for other smooth muscles because global elevation of cGMP after activation of soluble guanylyl cyclase does not relax the muscle. S-Nitrosylation, the covalent addition of an nitric oxide (NO) group to a cysteine thiol is a likely mechanism to explain the ability of NO to relax myometrium. This work is the first to describe the myometrial S-nitrosylproteome in both pregnant and nonpregnant tissue states. Using the guinea pig model, we show that specific sets of proteins involved in contraction and relaxation are S-nitrosylated in laboring and nonlaboring muscle and that many of these proteins are uniquely S-nitrosylated in only one state of the tissue. In particular, we show that S-nitrosylation of the intermediate filament protein desmin is significantly increased (5.7-fold, p < 0.005) in pregnancy and that this increase cannot be attributed solely to the increase in protein expression (1.8-fold, p < 0.005) that accompanies pregnancy. Elucidation of the myometrial S-nitrosylproteome provides a list of mechanistically important proteins that can constitute the basis of hypotheses formed to explain the regulation of uterine contraction/relaxation.

Fig. 1.

Representative schematic diagram of the biotin switch and the fluorescent switch. Proteins that have been previously S-nitrosylated can be either labeled with biotin-HPDP or an Alexa Fluor-maleimide dye. Step 1 involves blocking of all free thiols with either methyl methanethiosulfonate (MMTS) for the biotin switch or NEM for the fluorescent switch. Step 2 involves the reduction of all S-nitrosylated thiols with ascorbate. Reduced thiols, which were originally S-nitrosylated, are then labeled with biotin-HPDP or an Alexa Fluor-maleimide dye. The Alexa Fluor dye in step 3 of the fluorescent switch can also be 555 or 647 maleimide dyes.

Fig. 2.

Nitrosyl-DIGE analysis of nonpregnant and pregnant guinea pig USM. A representative nitrosyl-DIGE showing individual channels (red fluor, pregnant; green fluor, nonpregnant) as well as the two-dye overlay. Numbered spots were cut, trypsin-digested, and identified by MS analysis. 1, transitional endoplasmic reticulum ATPase; 2, serotransferrin; 3, vimentin; 4, desmin; 5, actin; 6, HSP27; 7, transgelin.

Fig. 3.

Comparison of increased levels of total desmin protein versus total levels of S-nitrosylated desmin. A, total desmin protein level from nonpregnant (NP) and pregnant (P) USM was separated by SDS-polyacrylamide gel electrophoresis, and a Western blot was performed using a desmin-specific antibody. Total desmin was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the fold increase was calculated to be 1.792. B, total desmin S-nitrosylation was determined from three independent nitrosyl-DIGE experiments using DeCyder software, and the fold increase was calculated to be 5.733. A Student's t test was performed to analyze the difference in means between groups, and the result was shown to be statistically significant with p = 0.0005. MW, molecular weight.