Uterine artery (UA) hydrogen sulfide (H2S) production is augmented in pregnancy and, on stimulation by systemic/local vasodilators, contributes to pregnancy-dependent uterine vasodilation; however, how H2S exploits this role is largely unknown. S-sulfhydration converts free thiols to persulfides at reactive cysteine(s) on targeted proteins to affect the entire proteome posttranslationally, representing the main route for H2S to elicit its function. Here, we used Tag-Switch to quantify changes in sulfhydrated (SSH-) proteins (ie, sulfhydrome) in H2S-treated nonpregnant and pregnant human UA. We further used the low-pH quantitative thiol reactivity profiling platform by which paired sulfhydromes were subjected to liquid chromatography tandem mass spectrometry-based peptide sequencing to generate site (cysteine)-specific pregnancy-dependent H2S-responsive human UA sulfhydrome. Total levels of sulfhydrated proteins were significantly greater in pregnant vs nonpregnant human UA and further stimulated by treatment with sodium hydrosulfide. We identified a total of 360 and 1671 SSH-peptides from 480 and 1186 SSH-proteins in untreated and sodium hydrosulfide-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 quantitative thiol reactivity profiling 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.

Liver development is a highly complex process that is regulated by the orchestrated interplay of epigenetic regulators, transcription factors, and microRNAs (miRNAs). Owing to the lack of global in vivo targets of all miRNAs during liver development, the mechanisms underlying the dynamic control of hepatocyte differentiation by miRNAs remain elusive. Here, using Argonaute (Ago) high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) in the mouse liver at different developmental stages, we characterized massive Ago-binding RNAs and obtained a genome-wide map of liver miRNA-mRNA interactions. The dynamic changes of five clusters of miRNAs and their potential targets were identified to be differentially involved at specific stages, a dozen of high abundant miRNAs and their epigenetic regulation by super-enhancer were found during liver development. Remarkably, miR-122, a liver-specific and most abundant miRNA in newborn and adult livers, was found by its targetome and pathway reporter analyses to regulate the Hippo pathway, which is crucial for liver size control and homeostasis. Mechanistically, we further demonstrated that miR-122 negatively regulates the outcomes of the Hippo pathway transcription factor TEAD by directly targeting a number of hippo pathway regulators, including the coactivator TAZ and a key factor of the phosphatase complex PPP1CC, which contributes to the dephosphorylation of YAP, another coactivator downstream of the Hippo pathway. This study identifies for the first time the genome-wide miRNA targetomes during mouse liver development and demonstrates a novel mechanism of terminal differentiation of hepatocytes regulated by the miR-122/Hippo pathway in a coordinated manner. As the Hippo pathway plays important roles in cell proliferation and liver pathological processes like inflammation, fibrosis, and hepatocellular carcinoma (HCC), our study could also provide a new insight into the function of miR-122 in liver pathology.