Vasopressin regulates transport across the collecting duct epithelium in part via effects on gene transcription. Transcriptional regulation occurs partially via changes in phosphorylation of transcription factors, transcriptional coactivators, and protein kinases in the nucleus. To test whether vasopressin alters the nuclear phosphoproteome of vasopressin-sensitive cultured mouse mpkCCD cells, we used stable isotope labeling and mass spectrometry to quantify thousands of phosphorylation sites in nuclear extracts and nuclear pellet fractions. Measurements were made in the presence and absence of the vasopressin analog dDAVP. Of the 1,251 sites quantified, 39 changed significantly in response to dDAVP. Network analysis of the regulated proteins revealed two major clusters ("cell-cell adhesion" and "transcriptional regulation") that were connected to known elements of the vasopressin signaling pathway. The hub proteins for these two clusters were the transcriptional coactivator β-catenin and the transcription factor c-Jun. Phosphorylation of β-catenin at Ser552 was increased by dDAVP [log(2)(dDAVP/vehicle) = 1.79], and phosphorylation of c-Jun at Ser73 was decreased [log(2)(dDAVP/vehicle) = -0.53]. The β-catenin site is known to be targeted by either protein kinase A or Akt, both of which are activated in response to vasopressin. The c-Jun site is a canonical target for the MAP kinase Jnk2, which is downregulated in response to vasopressin in the collecting duct. The data support the idea that vasopressin-mediated control of transcription in collecting duct cells involves selective changes in the nuclear phosphoproteome. All data are available to users at http://helixweb.nih.gov/ESBL/Database/mNPPD/.

Anemia is common among young children infected with Plasmodium falciparum (Pf) and severe malarial anemia (SMA) is a major cause of their mortality. Two major mechanisms cause malarial anemia: hemolysis of uninfected as well as infected erythrocytes and insufficient erythropoiesis. In a longitudinal birth cohort in Mali, we commonly observed marked hemoglobin reductions during Pf infections with a small proportion that progressed to SMA. We sought biomarkers of these processes using quantitative proteomic analysis on plasma samples from 9 P. falciparum-infected children, comparing those with reduced hemoglobin (with or without SMA) versus those with stable hemoglobin. We identified higher plasma levels of circulating 20S proteasome and lower IGF-1 levels in children with reduced hemoglobin. We confirmed these findings in independent ELISA-based validation studies of subsets of children from the same cohort (20S proteasome, N=71; IGF-1, N=78). We speculate that circulating 20S proteasome plays a role in digesting erythrocyte membrane proteins modified by oxidative stress, resulting in hemolysis, while decreased IGF-1, a critical factor for erythroid maturation, might contribute to insufficient erythropoiesis. Quantitative plasma proteomics identified soluble mediators that may contribute to the major mechanisms underlying malarial anemia.

Plasmodium falciparum variant antigens named erythrocyte membrane protein 1 (PfEMP1) are important targets for developing a protective immunity to malaria caused by P. falciparum. One of the major challenges in P. falciparum proteomics studies is identifying PfEMP1s at the protein level due to antigenic variation. To identify these PfEMP1s using shotgun proteomics, we developed a pipeline that searches high-resolution mass spectrometry spectra against a custom protein sequence database. A local alignment algorithm, LAX, was developed as a part of the pipeline that matches peptide sequences to the most similar PfEMP1 and calculates a weight value based on peptide's uniqueness used for PfEMP1 protein inference. The pipeline was first validated in the analysis of a laboratory strain with a known PfEMP1, then it was implemented on the analysis of parasite isolates from malaria-infected pregnant women and finally on the analysis of parasite isolates from malaria-infected children where there was an increase of PfEMP1s identified in 27 out of 31 isolates using the expanded database.