A novel PIP2 binding of epsilonPKC and its contribution to the neurite induction ability

Protein kinase C-epsilon (epsilonPKC) induces neurite outgrowth in neuroblastoma cells but molecular mechanism of the epsilonPKC-induced neurite outgrowth is not fully understood. Therefore, we investigated the ability of phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding of epsilonPKC and its correlation with the neurite extension. We found that full length epsilonPKC bound to PIP(2) in a 12-omicron-tetradecanoylphorbol-13-acetate dependent manner, while the regulatory domain of epsilonPKC (epsilonRD) bound to PIP(2) without any stimulation. To identify the PIP(2) binding region, we made mutants lacking several regions from epsilonRD, and examined their PIP(2) binding activity. The mutants lacking variable region 1 (V1) bound to PIP(2) stronger than intact epsilonRD, while the mutants lacking pseudo-substrate or common region 1 (C1) lost the binding. The PIP(2) binding ability of the V3-deleted mutant was weakened. Those PIP(2) bindings of epsilonPKC, epsilonRD and the mutants well correlated to their neurite induction ability. In addition, a chimera of pleckstrin homology domain of phospholipase Cdelta and the V3 region of epsilonPKC revealed that PIP(2) binding domain and the V3 region are sufficient for the neurite induction, and a first 16 amino acids in the V3 region was important for neurite extension. In conclusion, epsilonPKC directly binds to PIP(2) mainly through pseudo-substrate and common region 1, contributing to the neurite induction activity.     

Asexual growth of Babesia bovis is inhibited by specific sulfated glycoconjugates

In the present study, inhibitory effects of several sulfated and nonsulfated glycoconjugates were evaluated on the in vitro asexual growth of Babesia bovis. Among the selected sulfated glycoconjugates, dextran sulfate, heparin, heparan sulfate, fucoidan, and chondroitin sulfate B strongly inhibited the parasitic growth, and all but chondroitin sulfate B induced a significant accumulation of extracellular merozoites in culture. In contrast, chondroitin sulfate A, keratan sulfate, and protamine sulfate, as well as nonsulfated dextran and hyaluronic acid, did not influence the growth. These findings indicate that the asexual growth of B. bovis merozoites is inhibited by specific sulfated glycoconjugates, possibly providing us with an important insight into the molecular interaction(or interactions) during the process of the erythrocyte invasion by B. bovis merozoites.     

Rational design of a nonpeptide general chemical scaffold for reversible inhibition of PDZ domain interactions

Novel small molecules were designed to specifically target the ligand-binding pocket of a PDZ domain. Iterative molecular docking and modeling allowed the design of an indole scaffold 10a as a reversible inhibitor of ligand binding. The 10a scaffold inhibited the interaction between MAGI-3 and PTEN and showed cellular activities that are consistent with the inhibition of NHERF-1 function.     

Both the C1 domain and a basic amino acid cluster at the C-terminus are important for the neurite and branch induction ability of DGKβ

We previously reported that diacylglycerol kinase β (DGKβ) induces neurites and branches, contributing to higher brain function including emotion and memories. However, the detailed molecular mechanism of DGKβ function remains unknown. Therefore, we constructed various mutants of DGKβ and compared their enzyme activity, intracellular localization, and ability to induce neurites and branching in SH-SY5Y cells.     

A Small Molecule Inhibitor of Monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA) Inhibits Repair of Interstrand DNA Cross-link,Enhances DNA Double Strand Break,and Sensitizes Cancer Cells to Cisplatin

Small molecule inhibitors of proliferating cell nuclear antigen (PCNA)/PCNA interacting protein box (PIP-Box) interactions, including T2 amino alcohol (T2AA), inhibit translesion DNA synthesis. The crystal structure of PCNA in complex with T2AA revealed that T2AA bound to the surface adjacent to the subunit interface of the homotrimer of PCNA in addition to the PIP-box binding cavity. Because this site is close to Lys-164, which is monoubiquitinated by RAD18, we postulated that T2AA would affect monoubiquitinated PCNA interactions. Binding of monoubiquitinated PCNA and a purified pol η fragment containing the UBZ and PIP-box was inhibited by T2AA in vitro. T2AA decreased PCNA/pol η and PCNA/REV1 chromatin colocalization but did not inhibit PCNA monoubiquitination, suggesting that T2AA hinders interactions of pol η and REV1 with monoubiquitinated PCNA. Interstrand DNA cross-links (ICLs) are repaired by mechanisms using translesion DNA synthesis that is regulated by monoubiquitinated PCNA. T2AA significantly delayed reactivation of a reporter plasmid containing an ICL. Neutral comet analysis of cells receiving T2AA in addition to cisplatin revealed that T2AA significantly enhanced formation of DNA double strand breaks (DSBs) by cisplatin. T2AA promoted colocalized foci formation of phospho-ATM and 53BP1 and up-regulated phospho-BRCA1 in cisplatin-treated cells, suggesting that T2AA increases DSBs. When cells were treated by cisplatin and T2AA, their clonogenic survival was significantly less than that of those treated by cisplatin only. These findings show that the inhibitors of monoubiquitinated PCNA chemosensitize cells by inhibiting repair of ICLs and DSBs.     

Investigation of the PDZ domain ligand binding site using chemically modified peptides

Several chemically modified analogues to a tightly binding ligand for the second PDZ domain of MAGI-3 were synthesized and evaluated for their ability to compete with native peptide ligands. N-methyl scanning of the ligand backbone amides revealed the energetically important hydrogen bonds between the ligand backbone and the PDZ domain. Analogues to the ligand's conserved threonine/serine(-2) residue, involved in a side chain to side chain hydrogen bond with a conserved histidine in the PDZ domain, revealed that the interaction is highly sensitive to the steric structure around the hydroxyl group of this residue. Analogues of the ligand carboxy terminus revealed that the full hydrogen bond network of the GLGF loop is important in ligand binding.     

Oxidative Stress Induced Inflammation Initiates Functional Decline of Tear Production

Oxidative damage and inflammation are proposed to be involved in an age-related functional decline of exocrine glands. However, the molecular mechanism of how oxidative stress affects the secretory function of exocrine glands is unclear. We developed a novel mev-1 conditional transgenic mouse model (Tet-mev-1) using a modified tetracycline system (Tet-On/Off system). This mouse model demonstrated decreased tear production with morphological changes including leukocytic infiltration and fibrosis. We found that the mev-1 gene encodes Cyt-1, which is the cytochrome b₅₆₀ large subunit of succinate-ubiquinone oxidoreductase in complex II of mitochondria (homologous to succinate dehydrogenase C subunit (SDHC) in humans). The mev-1 gene induced excessive oxidative stress associated with ocular surface epithelial damage and a decrease in protein and aqueous secretory function. This new model provides evidence that mitochondrial oxidative damage in the lacrimal gland induces lacrimal dysfunction resulting in dry eye disease. Tear volume in Tet-mev-1 mice was lower than in wild type mice and histopathological analyses showed the hallmarks of lacrimal gland inflammation by intense mononuclear leukocytic infiltration and fibrosis in the lacrimal gland of Tet-mev-1 mice. These findings strongly suggest that oxidative stress can be a causative factor for the development of dry eye disease.     

A novel putative lipoprotein receptor (CasLpR) in the hemocytes of the blue crab, Callinectes sapidus: cloning and up-regulated expression after the injection of LPS and LTA

The full-length cDNA encoding a putative lipoprotein receptor (CasLpR) was isolated from the hemocytes of Callinectes sapidus using 5′ and 3′ RACEs. The open reading frame for CasLpR contains a precursor of putative CasLpR consisting of 1710 amino acid residues including 22 amino acid residues of the signal peptide (22 amino acids). Mature CasLpR (1688 amino acids with 5.6% of phosphorylation sites) has multiple, putative functional domains: five low-density lipoprotein receptor domains in the N-terminus, and a G-protein-coupled receptor proteolysis site domain and a 7 transmembrane receptor (secretin family) domain in the C-terminus. To date, there are no proteins with a similar domain structure in the GenBank. The expression pattern of CasLpR was exclusive in hemocytes among all tested tissues obtained from a juvenile female at intermolt stage: brain, eyestalk ganglia, pericardial organs, and thoracic ganglia complex (nervous system); hepatopancreas (digestive system); heart, artery and hemocytes (circulatory system); gill and antennal gland (excretory system), hypodermis; and Y-organ (endocrine organ). There was no CasLpR expression in the ovary of an adult female. A putative function of CasLpR was examined after challenges of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) in vivo using qRT-PCR assays. Animals at 24 h after injection of LPS or LTA up-regulated the expression of CasLpR in hemocytes by ∼3.5 and 1.4 folds, respectively, compared to the controls that received saline injection. LPS challenge also caused the greatest increment (∼55 folds) of heat shock protein 90 (Hsp90) expression in these samples. These data indicate that putative CasLpR and CasHsp90 may be involved in the defense system or the stress response of C. sapidus.     

Gliding motility of Babesia bovis merozoites visualized by time-lapse video microscopy

Background

Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed “gliding motility”. However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite.

Methodology/Principal Findings

Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion.

Conclusions/Significance

This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding.