Intimations on the Regulation of Myometrial Functions: Lessons from the Proteome and S-nitrosoproteome in Human Preterm Labor
AuthorUlrich, Craig Corwin
AdvisorBuxton, Iain L.O.
Biochemistry and Molecular Biology
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The molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor are not completely known. More than 10% of babies born worldwide are premature and one million die each year. Preterm labor results in preterm delivery in 50% of cases in the US explaining 75% of fetal morbidity and mortality. There is no FDA-approved treatment available to prevent preterm delivery. Nitric oxide relaxes uterine smooth muscle in a manner disparate from other smooth muscles since global elevation of cGMP following activation of soluble guanylyl cyclase does not relax the muscle. S-nitrosation, the covalent addition of an 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-nitrosoproteome in both the pregnant and non-pregnant tissue states. Using the guinea pig model, we show that specific sets of proteins involved in contraction and relaxation are S-nitrosated in laboring and non-laboring muscle and that many of these proteins are uniquely S-nitrosated in only one state of the tissue. In particular, we show that S-nitrosation 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-nitrosoproteome provides a list of mechanistically important proteins that can constitute the basis of hypotheses formed to explain the regulation of uterine contraction-relaxation.Identification and semi-quantification of proteomic changes during pregnancy will allow for targeted research into how the induction of labor occurs. We have recently performed two dimensional liquid chromatography coupled with tandem mass spectrometry on myometrial proteins isolated from pregnant human patients in labor, pregnant patients not in labor, and pregnant patients in pre-term labor. Using a conservative false discovery rate of 1% we have identified 2132 protein groups using this method and semi-quantitative spectral counting shows 201 proteins that have disparate levels of protein expression in the preterm laboring samples. To our knowledge this is the first large scale proteomic project on human uterine smooth muscle and this initial work has provided a target list for future experiments into how changing levels of proteins are involved in the induction of labor.Employing S-nitrosoglutathione as a nitric oxide donor, we identified 110 proteins that are S-nitrosated in one or more states of human pregnancy. Using area under the curve of extracted ion chromatograms as well as normalized spectral counts to quantify relative expression levels for 62 of these proteins, we show that 26 proteins demonstrate statistically significant differences in myometrium from spontaneously laboring preterm patients and non-laboring patients. We identified proteins that were up-S-nitrosated as well as proteins that were down-S-nitrosated in preterm laboring tissues. Identification and relative quantification of the S-nitrosoproteome provides a fingerprint of mechanistically important proteins that can form the basis of hypothesis-directed efforts to understand the regulation of uterine contraction-relaxation and the development of new treatment for preterm labor.