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:Androgen receptor (AR)-targeting prostate cancer drugs, which are predominantly competitive ligand binding domain (LBD)-binding antagonists, are inactivated by common resistance -mechanisms. It is important to develop next-generation mechanistically-distinct drugs to treat castration- and drug- resistant prostate cancers. Here, we describe a second-generation AR pan-antagonist (UT-34) that degrades the AR and AR splice variants. UT-34 inhibits the wild-type and LBD mutant ARs comparably and inhibits the in vitro proliferation and in vivo growth of enzalutamide-sensitive and resistant prostate cancer xenografts. In preclinical models, UT-34 induced the regression of enzalutamide-resistant tumors at doses when the AR is degraded; but, at lower doses when the AR is just antagonized, it inhibits, without shrinking, the tumors. This indicates that degradation might be a prerequisite for tumor regression. Mechanistically, UT-34 promotes a conformation that is distinct from the LBD-binding competitive antagonist, enzalutamide, and degrades the AR through the ubiquitin proteasome mechanism. UT-34 has a broad safety margin and exhibits no cross-reactivity with G-Protein Coupled Receptor, kinase, and nuclear receptor family members. Collectively, UT-34 exhibits the properties necessary for a next-generation prostate cancer drug. MR49F cells (n=3-4/group) were maintained in charcoal-stripped serum containing medium for 48 hours and treated with vehicle, 0.1 nM R1881, or 10 uM of UT-34 in combination with 0.1 nM R1881. Twenty four hours after treatment, the cells were harvested, RNA was isolated and expression of genes was measured using microarray (Affymetrix Clarion S) UT-34 is a selective androgen receptor degrader that degrades and antagonizes the AR. UT-34 binds to the AF-1 domain of the AR and degrades the AR through ubiquitin proteasome pathways. The transcriptome study was performed to evaluate the ability of UT-34 to antagonize the enzalutamide-resistant AR function.
Project description:In this study we analyze the in vivo recruitment of mouse brain nuclear Rbfox proteins to RNA using individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP). Rbfox1, Rbfox, 2, and Rbfox3 iCLIP libraries were constructed from high molecular weight (HMW) nuclear fraction containing chromatin and the soluble nucleoplasm.