Project description:The urea channel Slc14a2 (or UT-A1) mediates vasopressin-regulated urea transport across the inner medullary collecting duct (IMCD). Previously, UT-A1 was found to present in a high molecular weight complex, suggesting UT-A1 is involved in certain protein-protein interactions. The present study sought to identify the proteins that interact with UT-A1 in this complex for a better understanding of how UT-A1 is regulated. Rat IMCD suspensions were treated with or without V2 receptor agonist, dDAVP, followed by in-cell crosslinking using BSOCOES and detergent solubilization. Immunoprecipitation using Dynabeads coated with UT-A1 specific antibody successfully pulled down the UT-A1 proteins. In-gel digestion protocol was carried out to prepare samples for liquid chromatographic mass spectrometry analysis of tryptic peptides using a Velos-Orbitrap mass spectrometer. The peptides passing stringent spectral quality thresholds were quantified (label-free) to identify those with (UTA-1 antibody/preimmune IgG) >4. A total of 128 UT-A1 interacting proteins were identified. Gene Ontology analysis maps the distribution of these proteins throughout major cell compartments: endoplasmic reticulum, Golgi, endosomes, cytosol and plasma membrane. Among them are four protein kinases (Cdc42bpb, Phkb, Camk2d, Mtor) that play roles in vasopressin-regulated phosphorylation of UT-A1. Non-label quantification was also performed to determine the stoichiometry of UT-A3 with UT-A1, the result does not support an oligomeric complex formation of UT-A1/A3. In conclusion, we have provided a refined list of UT-A1 binding proteins which can be useful for further analysis of the vasopressin signaling pathway in regulation of UT-A1 in IMCD.
Project description:An important question for the use of the mouse as a model for studying human disease is the degree of functional conservation of genetic control pathways from human to mouse. The human and mouse placenta show structural similarities but there have been no systematic attempt to assess their molecular similarities or differences. We built a comprehensive database of protein and microarray data for the highly vascular exchange region micro-dissected from the human and mouse placenta near-term. Abnormalities in this region are associated with two of the most common and serious complications of human pregnancy, maternal preeclampsia (PE) and fetal intrauterine growth restriction (IUGR), each disorder affecting ~5% of all pregnancies. Over 7,000 orthologs were detected with 70% co-expressed and over 80% of genes known to cause placental phenotypes in mouse were co-expressed. These genes form a tight protein-protein interaction network with novel candidate genes likely to be important in placental structure and/or function. The entire data is available as a web-accessible database to guide the informed development of mouse models to study human disease This experiment is now fully represented in NCBI Peptidome database with accession PSE115; http://www.ncbi.nlm.nih.gov/peptidome/search/index.shtml?acc=PSE115 Microdissection of human villous trees and mouse placental labyrinth. Tissues were split for microarray and protein analysis. For protein analysis samples were first fractionated by differential sucrose gradients into mitochrondria, cytosol, microsomes and nuclei. Mitochrondira and neuclei were each extracted by two different methods for soluble and insoluble material. Each subcellular fraction for each tissue was analysed in quintuplet by 9 step 2 dimensional LC/MSMS. This generated a total of 270 mzXML files for each tissue.