Mapping Pregnancy-dependent Sulfhydrome Unfolds Diverse Functions of Protein Sulfhydration in Human Uterine Artery.

Abstract

Uterine artery hydrogen sulfide (H2S) production is augmented in pregnancy and upon stimulation by systemic/local vasodilators, contributing to pregnancy-dependent uterine vasodilation; however, how H2S exploits this role is largely unknown. S-sulfhydration converts free thiols (-SH) to persulfides (-SSH) at reactive cysteine(s) on targeted proteins to affect the entire proteome post-translationally, representing the main route for H2S to elicit its function. Herein we used Tag-Switch to quantify changes in sulfhydrated (SSH-) proteins (i.e., sulfhydromes) in H2S-treated nonpregnant (NP) and pregnant (P) human UA. We further used the Low-pH quantitative thiol reactivity profiling (low-pH QTRP) platform by which paired sulfhydromes were subjected to LC-MS/MS based peptide sequencing to generate site (cysteine)-specific pregnancy-dependent H2S-responsive human UA sulfhydrome. Total levels of sulfhydrated proteins were significantly greater in P versus NP human UA and further stimulated by treatment with NaHS. We identified a total of 360 and 1,671 SSH-peptides from 480 and 1,186 SSH-proteins in untreated and NaHS-treated human UA, respectively. Bioinformatics analyses identified pregnancy-dependent H2S-responsive human UA SSH-peptides/proteins, which were categorized to various molecular functions, pathways, and biological processes, especially vascular smooth muscle contraction/relaxation. Pregnancy-dependent changes in these proteins were rectified by immunoblotting of the Tag-Switch labeled SSH-proteins. Low-pH QTRP failed to identify low abundance SSH-proteins such as KATP channels in human UA; however, immunoblotting of Tag-Switch labeled SSH-proteins identified pregnancy-dependent upregulation of SSH-KATP channels without altering their total proteins. Thus, comprehensive analyses of human UA sulfhydromes influenced by endogenous and exogenous H2S inform novel roles of protein sulfhydration in uterine hemodynamics regulation.