BackgroundPredicting drug-protein interactions from heterogeneous biological data sources is a key step for in silico drug discovery. The difficulty of this prediction task lies in the rarity of known drug-protein interactions and myriad unknown interactions to be predicted. To meet this challenge, a manifold regularization semi-supervised learning method is presented to tackle this issue by using labeled and unlabeled information which often generates better results than using the labeled data alone. Furthermore, our semi-supervised learning method integrates known drug-protein interaction network information as well as chemical structure and genomic sequence data.ResultsUsing the proposed method, we predicted certain drug-protein interactions on the enzyme, ion channel, GPCRs, and nuclear receptor data sets. Some of them are confirmed by the latest publicly available drug targets databases such as KEGG.ConclusionsWe report encouraging results of using our method for drug-protein interaction network reconstruction which may shed light on the molecular interaction inference and new uses of marketed drugs.

Gain/loss of function studies were utilized to assess the potential role of the endogenous vanilloid receptor TRPV4 as a sensor of flow and osmolality in M-1 collecting duct cells (CCD). TRPV4 mRNA and protein were detectable in M-1 cells and stably transfected HEK-293 cells, where the protein occurred as a glycosylated doublet on Western blots. Immunofluorescence imaging demonstrated expression of TRPV4 at the cell membranes of TRPV4-transfected HEK and M-1 cells and at the luminal membrane of mouse kidney CCD. By using intracellular calcium imaging techniques, calcium influx was monitored in cells grown on coverslips. Application of known activators of TRPV4, including 4alpha-PDD and hypotonic medium, induced strong calcium influx in M-1 cells and TRPV4-transfected HEK-293 cells but not in nontransfected cells. Applying increased flow/shear stress in a parallel plate chamber induced calcium influx in both M-1 and TRPV4-transfected HEK cells but not in nontransfected HEK cells. Furthermore, in loss-of-function studies employing small interference (si)RNA knockdown techniques, transfection of both M-1 and TRPV4-transfected HEK cells with siRNA specific for TRPV4, but not an inappropriate siRNA, led to a time-dependent decrease in TRPV4 expression that was accompanied by a loss of stimuli-induced calcium influx to flow and hypotonicity. It is concluded that TRPV4 displays a mechanosensitive nature with activation properties consistent with a molecular sensor of both fluid flow (or shear stress) and osmolality, or a component of a sensor complex, in flow-sensitive renal CCD.

The preliminary data clearly demonstrate a rapid uptake of 2-NBDG into tumor cells that can be monitored by fluorescence imaging analysis. The uptake displays saturation and competition with D -glucose, all properties expected for 2-NBDG uptake and retention in cancer cells. Additional studies, including comparisons among other malignant cell lines and control cells, will be needed to fully characterize the kinetic properties of 2-NBDG uptake and the potential use of this 2-DG analog as a probe for glucose uptake in malignant cells.

The four R-spondins (RSPO1-4) strongly potentiate Wnt signaling and play critical roles in normal development, adult stem cell survival, and cancer development and aggressiveness. All four RSPOs have been suggested to potentiate Wnt signaling by binding to three related receptors, leucine-rich repeat-containing, G protein-coupled receptors 4, 5, and 6 (LGR4/5/6), and then inducing the clearance of two E3 ubiquitin ligases (RNF43 and ZNRF3) that otherwise would ubiquitinate Wnt receptors for degradation. Here, we show that RSPO1-4 have differential dependence on LGRs in potentiating Wnt/β-catenin signaling and that RSPO2 can enhance this pathway without any LGR. LGR4 knockout (LGR4KO) in HEK293 cells completely abrogated the Wnt/β-catenin signaling response to RSPO1 and RSPO4 and strongly impaired the response to RSPO3. RSPO2, however, retained robust activity albeit with decreased potency. Complete rescue of RSPO1-4 activity in LGR4KO cells required the seven-transmembrane domain of LGR4. Furthermore, an RSPO2 mutant with normal binding affinity to ZNRF3 but no or little binding to LGR4 or LGR5 still potentiated Wnt/β-catenin signaling, supported the growth of intestinal organoids , and stimulated intestinal crypt growth Mechanistically, RSPO2 could increase Wnt receptor levels in the absence of any LGR without affecting ZNRF3 endocytosis and stability. These findings suggest that RSPO1-4 use distinct mechanisms in regulating Wnt and other signaling pathways, which have important implications for understanding the pleiotropic functions of RSPOs and LGRs in both normal and cancer development.