Tyrosine phosphorylation enhances RAD52-mediated annealing by modulating its DNA binding

RAD52 protein has an important role in homology-directed DNA repair by mediating RAD51 nucleoprotein filament formation on single-stranded DNA (ssDNA) protected by replication protein-A (RPA) and annealing of RPA-coated ssDNA. In human, cellular response to DNA damage includes phosphorylation of RAD52 by c-ABL kinase at tyrosine 104. To address how this phosphorylation modulates RAD52 function, we used an amber suppressor technology to substitute tyrosine 104 with chemically stable phosphotyrosine analogue (p-Carboxymethyl-L-phenylalanine, pCMF). The RAD52(Y104pCMF) retained ssDNA-binding activity characteristic of unmodified RAD52 but showed lower affinity for double-stranded DNA (dsDNA) binding. Single-molecule analyses revealed that RAD52(Y104pCMF) specifically targets and wraps ssDNA. While RAD52(Y104pCMF) is confined to ssDNA region, unmodified RAD52 readily diffuses into dsDNA region. The Y104pCMF substitution also increased the ssDNA annealing rate and allowed overcoming the inhibitory effect of dsDNA. We propose that phosphorylation at Y104 enhances ssDNA annealing activity of RAD52 by attenuating dsDNA binding. Implications of phosphorylation-mediated activation of RAD52 annealing activity are discussed.     

The role of L1 loop in the mechanism of rhomboid intramembrane protease GlpG

Intramembrane proteases are important enzymes in biology. The recently solved crystal structures of rhomboid protease GlpG have provided useful insights into the mechanism of these membrane proteins. Besides revealing an internal water-filled cavity that harbored the Ser-His catalytic dyad, the crystal structure identified a novel structural domain (L1 loop) that lies on the side of the transmembrane helices. Here, using site-directed mutagenesis, we confirmed that the L1 loop is partially embedded in the membrane, and showed that alanine substitution of a highly preferred tryptophan (Trp136) at the distal tip of the L1 loop near the lipid:water interface reduced GlpG proteolytic activity. Crystallographic analysis showed that W136A mutation did not modify the structure of the protease. Instead, the polarity for a small and lipid-exposed protein surface at the site of the mutation has changed. The crystal structure, now refined at 1.7 Å resolution, also clearly defined a 20-Å-wide hydrophobic belt around the protease, which likely corresponded to the thickness of the compressed membrane bilayer around the protein. This improved structural model predicts that all critical elements of the catalysis, including the catalytic serine and the L5 cap, need to be positioned within a few angstroms of the membrane surface, and may explain why the protease activity is sensitive to changes in the protein:lipid interaction. Based on these findings, we propose a model where the end of the substrate transmembrane helix first partitions out of the hydrophobic core region of the membrane before it bends into the protease active site for cleavage.     

BPR1K653, a novel Aurora kinase inhibitor, exhibits potent anti-proliferative activity in MDR1 (P-gp170)-mediated multidrug-resistant cancer cells

Background

Over-expression of Aurora kinases promotes the tumorigenesis of cells. The aim of this study was to determine the preclinical profile of a novel pan-Aurora kinase inhibitor, BPR1K653, as a candidate for anti-cancer therapy. Since expression of the drug efflux pump, MDR1, reduces the effectiveness of various chemotherapeutic compounds in human cancers, this study also aimed to determine whether the potency of BPR1K653 could be affected by the expression of MDR1 in cancer cells.

Principal Findings

BPR1K653 specifically inhibited the activity of Aurora-A and Aurora-B kinase at low nano-molar concentrations in vitro. Anti-proliferative activity of BPR1K653 was evaluated in various human cancer cell lines. Results of the clonogenic assay showed that BPR1K653 was potent in targeting a variety of cancer cell lines regardless of the tissue origin, p53 status, or expression of MDR1. At the cellular level, BPR1K653 induced endo-replication and subsequent apoptosis in both MDR1-negative and MDR1-positive cancer cells. Importantly, it showed potent activity against the growth of xenograft tumors of the human cervical carcinoma KB and KB-derived MDR1-positive KB-VIN10 cells in nude mice. Finally, BPR1K653 also exhibited favorable pharmacokinetic properties in rats.

Conclusions and Significance

BPR1K653 is a novel potent anti-cancer compound, and its potency is not affected by the expression of the multiple drug resistant protein, MDR1, in cancer cells. Therefore, BPR1K653 is a promising anti-cancer compound that has potential for the management of various malignancies, particularly for patients with MDR1-related drug resistance after prolonged chemotherapeutic treatments.     

Crystal structure and thermostability of a putative α-glucosidase from Thermotoga neapolitana

Glycoside hydrolase family 4 (GH4) represents an unusual group of glucosidases with a requirement for NAD(+), Mn(2+), and reducing conditions. We found a putative α-glucosidase belonging to GH4 in hyperthermophilic Gram-negative bacterium Thermotoga neapolitana. In this study, we recombinantly expressed the putative α-glycosidase from T. neapolitana, and determined the crystal structure of the protein at a resolution of 2.0? in the presence of Mn(2+) but in the absence of NAD(+). The structure showed the dimeric assembly and the Mn(2+) coordination that other GH4 enzymes share. In comparison, we observed structural changes in T. neapolitana α-glucosidase by the binding of NAD(+), which also increased the thermostability. Numerous arginine-mediated salt-bridges were observed in the structure, and we confirmed that the salt bridges correlated with the thermostability of the proteins. Disruption of the salt bridge that linked N-terminal and C-terminal parts at the surface dramatically decreased the thermostability. A mutation that changed the internal salt bridge to a hydrogen bond also decreased the thermostability of the protein. This study will help us to understand the function of the putative glucosidase and the structural features that affect the thermostability of the protein.     

Chromosome aberrations in workers of nuclear-power plants

The frequencies of chromosome aberrations in 135 workers from nuclear-power plants were compared with those in 135 age-matched controls. A total of 135,000 cells was scored. The frequencies of dicentric chromosome were 1.67 × 10⁻³ in the exposed group and 0.49 × 10⁻³ in the control group and those of chromosome-type deletion were 3.33 × 10⁻³ and 1.10 × 10⁻³, respectively. The frequencies of all types of chromosome aberrations in the exposed subjects were higher than those in the control group, but no significant trend of dose-dependent increase was observed when only the exposed group were considered. Poisson regression analysis, with both exposed and control included, showed that there was a significant association of chromosome aberration with radiation dose and the duration of work, but not with age, smoking habit and alcohol intake. It was also found that recent exposure to radiation, within the last 5 years, had contributed more to the observed chromosome aberration than earlier exposure.     

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study

1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP.     

Lipid-Based Nanocarriers via Nose-to-Brain Pathway for Central Nervous System Disorders

Neurochemical Research - Neurodegenerative disorders are distinguished by the gradual deterioration of the nervous system’s structure and function due to oxidative stress, mitochondrial...