N4WBP5, a potential target for ubiquitination by the Nedd4 family of proteins, is a novel Golgi-associated protein.

Nedd4 belongs to a family of ubiquitin-protein ligases that is characterized by 2--4 WW domains, a carboxyl-terminal Hect (homologous to E6-AP Carboxyl terminus)domain and in most cases an amino-terminal C2 domain. We had previously identified a series of proteins that associates with the WW domains of Nedd4. In this paper, we demonstrate that one of the Nedd4-binding proteins, N4WBP5, belongs to a small group of evolutionarily conserved proteins with three transmembrane domains. N4WBP5 binds Nedd4 WW domains via the two PPXY motifs present in the amino terminus of the protein. In addition to Nedd4, N4WBP5 can interact with the WW domains of a number of Nedd4 family members and is ubiquitinated. Endogenous N4WBP5 localizes to the Golgi complex. Ectopic expression of the protein disrupts the structure of the Golgi, suggesting that N4WBP5 forms part of a family of integral Golgi membrane proteins. Based on previous observations in yeast, we propose that N4WBP5 may act as an adaptor for Nedd4-like proteins and their putative targets to control ubiquitin-dependent protein sorting and trafficking.     

Novel 3-methylindoline inhibitors of EZH2: Design,synthesis and SAR

EZH2 (enhancer of zeste homologue 2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that catalyzes the methylation of lysine 27 of histone H3 (H3K27). Dysregulation of EZH2 activity is associated with several human cancers and therefore EZH2 inhibition has emerged as a promising therapeutic target. Several small molecule EZH2 inhibitors with different chemotypes have been reported in the literature, many of which use a bicyclic heteroaryl core. Herein, we report the design and synthesis of EZH2 inhibitors containing an indoline core. Partial saturation of an indole to an indoline provided lead compounds with nanomolar activity against EZH2, while also improving solubility and oxidative metabolic stability.     

Mathematical modeling of fission yeast Schizosaccharomyces pombe cell cycle: exploring the role of multiple phosphatases

Cell cycle is the central process that regulates growth and division in all eukaryotes. Based on the environmental condition sensed, the cell lies in a resting phase G0 or proceeds through the cyclic cell division process (G1??S??G2??M). These series of events and phase transitions are governed mainly by the highly conserved Cyclin dependent kinases (Cdks) and its positive and negative regulators. The cell cycle regulation of fission yeast Schizosaccharomyces pombe is modeled in this study. The study exploits a detailed molecular interaction map compiled based on the published model and experimental data. There are accumulating evidences about the prominent regulatory role of specific phosphatases in cell cycle regulations. The current study emphasizes the possible role of multiple phosphatases that governs the cell cycle regulation in fission yeast S. pombe. The ability of the model to reproduce the reported regulatory profile for the wild-type and various mutants was verified though simulations.     

The ubiquitin ligase NEDD4-2/NEDD4L regulates both sodium homeostasis and fibrotic signaling to prevent end-stage renal disease

Kidney disease progression can be affected by Na⁺ abundance. A key regulator of Na⁺ homeostasis is the ubiquitin ligase NEDD4-2 and its deficiency leads to increased Na⁺ transport activity and salt-sensitive progressive kidney damage. However, the mechanisms responsible for high Na⁺ induced damage remain poorly understood. Here we show that a high Na⁺ diet compromised kidney function in Nedd4-2-deficient mice, indicative of progression toward end-stage renal disease. Injury was characterized by enhanced tubule dilation and extracellular matrix accumulation, together with sustained activation of both Wnt/β-catenin and TGF-β signaling. Nedd4-2 knockout in cortical collecting duct cells also activated these pathways and led to epithelial–mesenchymal transition. Furthermore, low dietary Na⁺ rescued kidney disease in Nedd4-2-deficient mice and silenced Wnt/β-catenin and TGF-β signaling. Our study reveals the important role of NEDD4-2-dependent ubiquitination in Na⁺ homeostasis and protecting against aberrant Wnt/β-catenin/TGF-β signaling in progressive kidney disease.Subject terms: Stress signalling, End-stage renal disease     

Prediction of Epitope-Based Peptides for Vaccine Development from Coat Proteins GP2 and VP24 of Ebola Virus Using Immunoinformatics

This study aims to design epitope-based peptides for the utility of vaccine development by targeting Glycoprotein 2 (GP2) and Viral Protein 24 (VP24) of the Ebola virus (EBOV) that, respectively, facilitate attachment and fusion of EBOV with host cells. Using various databases and tools, immune parameters of conserved sequences from GP2 and VP24 proteins of different strains of EBOV were tested to predict probable epitopes. Binding analyses of the peptides with major histocompatibility complex (MHC) class I and class II molecules, population coverage, and linear B cell epitope prediction were peroformed. Predicted peptides interacted with multiple MHC alleles and illustrated maximal population coverage for both GP2 and VP24 proteins, respectively. The predicted class-I nonamers, FLYDRLAST, LFLRATTEL and NYNGLLSSI were found to cover the maximum number of MHC I alleles and showed interactions with binding energies of ?7.8, ?8.5 and ?7.7 kcal/mol respectively. Highest scoring class II MHC binding peptides were EGAFFLYDRLASTVI and SPLWALRVILAAGIQ with binding energies of ?6.2 and -5.6 kcal/mol. Putative B cell epitopes were also found on 4 conserved regions in GP2 and two conserved regions in VP24. Our in silico analysis suggests that the predicted epitopes could be a better choice as universal vaccine component against EBOV irrespective of different strains and should be subjected to in vitro and in vivo analyses for further research and development.     

Prioritising Healthcare Workers for Ebola Treatment: Treating Those at Greatest Risk to Confer Greatest Benefit

The Ebola epidemic in Western Africa has highlighted issues related to weak health systems, the politics of drug and vaccine development and the need for transparent and ethical criteria for use of scarce local and global resources during public health emergency. In this paper we explore two key themes. First, we argue that independent of any use of experimental drugs or vaccine interventions, simultaneous implementation of proven public health principles, community engagement and culturally sensitive communication are critical as these measures represent the most cost‐effective and fair utilization of available resources. Second, we attempt to clarify the ethical issues related to use of scarce experimental drugs or vaccines and explore in detail the most critical ethical question related to Ebola drug or vaccine distribution in the current outbreak: who among those infected or at risk should be prioritized to receive any new experimental drugs or vaccines? We conclude that healthcare workers should be prioritised for these experimental interventions, for a variety of reasons.     

Inhibition of liver metastases in murine colon adenocarcinoma by liposomes containing human C-reactive protein or crude lymphokine

Previous studies from our laboratory have shown that liposomes containing LYNK or CRP inhibit lung metastases in mice bearing the malignant fibrosarcoma, T241. We have now extended these observations to the murine colon adenocarcinoma (MCA-38), which metastasizes to the liver. MCA-38 tumor cells (1 X 10(6)) were implanted in the wall of the cecum by orthotopic transplantation. Three-hundred twenty-six mice bearing such tumors were divided into four treatment groups as follows: (1) no treatment (2) liposomes containing control medium (3) liposomes containing LYNK, and (4) liposomes containing CRP. Treatment was started from day 2, day 18, or 34 after tumor implantation and it was administered on 3 days per week. Each treatment, given parenterally, consisted of 4 mumol liposomes (PS-PC, 1:1) containing the appropriate agents. Mice receiving liposomes containing LYNK or CRP had significantly fewer and smaller liver metastases (25%-28%), than those in the two control groups (53%-54%). Also, a significantly better survival was noted in the two treated groups than in the two control groups. The most likely mechanism of tumor inhibition appears to be through macrophage activation. In the Winn tumor neutralization test, peritoneal macrophages harvested from normal mice 24 h after a single injection of liposomes (2.5 mumol) containing LYNK or CRP markedly inhibited tumor growth when a mixture of effector-target cells (40 : 1) was injected in the footpad. These studies further confirm the suggested role of CRP as an 'immunomodulator' or 'biological response modifier' in yet another tumor system.     

Phosphorylation by Aurora B kinase regulates caspase-2 activity and function

Mitotic catastrophe (MC) is an important oncosuppressive mechanism that serves to eliminate cells that become polyploid or aneuploid due to aberrant mitosis. Previous studies have demonstrated that the activation and catalytic function of caspase-2 are key steps in MC to trigger apoptosis and/or cell cycle arrest of mitotically defective cells. However, the molecular mechanisms that regulate caspase-2 activation and its function are unclear. Here, we identify six new phosphorylation sites in caspase-2 and show that a key mitotic kinase, Aurora B kinase (AURKB), phosphorylates caspase-2 at the highly conserved residue S384. We demonstrate that phosphorylation at S384 blocks caspase-2 catalytic activity and apoptosis function in response to mitotic insults, without affecting caspase-2 dimerisation. Moreover, molecular modelling suggests that phosphorylation at S384 may affect substrate binding by caspase-2. We propose that caspase-2 S384 phosphorylation by AURKB is a key mechanism that controls caspase-2 activation during mitosis.Subject terms: Proteases, Proteolysis