Hyperbranched Double Hydrophilic Block Copolymer Micelles of Poly(ethylene oxide) and Polyglycerol for pH-Responsive Drug Delivery

We report the synthesis of a well-defined hyperbranched double hydrophilic block copolymer of poly­(ethylene oxide)-<i>hyperbranched</i>-polyglycerol (PEO-<i>hb</i>-PG) to develop an efficient drug delivery system. In specific, we demonstrate the hyperbranched PEO-<i>hb</i>-PG can form a self-assembled micellar structure on conjugation with the hydrophobic anticancer agent doxorubicin, which is linked to the polymer by pH-sensitive hydrazone bonds, resulting in a pH-responsive controlled release of doxorubicin. Dynamic light scattering, atomic force microscopy, and transmission electron microscopy demonstrated successful formation of the spherical core–shell type micelles with an average size of about 200 nm. Moreover, the pH-responsive release of doxorubicin and in vitro cytotoxicity studies revealed the controlled stimuli-responsive drug delivery system desirable for enhanced efficiency. Benefiting from many desirable features of hyperbranched double hydrophilic block copolymers such as enhanced biocompatibility, increased water solubility, and drug loading efficiency as well as improved clearance of the polymer after drug release, we believe that double hydrophilic block copolymer will provide a versatile platform to develop excellent drug delivery systems for effective treatment of cancer

Inhibitory RNA Aptamers of Tau Oligomerization and Their Neuroprotective Roles against Proteotoxic Stress

Tau is a cytosolic protein that functions in the assembly and stabilization of axonal microtubule networks. Its oligomerization may be the rate-limiting step of insoluble aggregate formation, which is a neuropathological hallmark of Alzheimer’s disease (AD) and a number of other tauopathies. Recent evidence indicates that soluble tau oligomers are the toxic species for tau-mediated pathology during AD progression. Herein, we describe novel RNA aptamers that target human tau and were identified through an <i>in vitro</i> selection process. These aptamers significantly inhibited the oligomerization propensity of tau both <i>in vitro</i> and in cultured cell models of tauopathy without affecting the half-life of tau. Tauopathy model cells treated with the aptamers were less sensitized to proteotoxic stress induced by tau overexpression. Moreover, the tau aptamers significantly alleviated synthetic tau oligomer-mediated neurotoxicity and dendritic spine loss in primary hippocampal neurons. Thus, our study demonstrates that delaying tau assembly with RNA aptamers is an effective strategy for protecting cells under various neurodegenerative stresses originating from pathogenic tau oligomerization

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

<p>The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using <i>para</i>-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, <i>ATE1</i>-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux.</p

Antibodies used in immunohistochemistry.

<p>Antibodies used in immunohistochemistry.</p

Total ubiquitylation activities are significantly reduced in <i>UBR2^−/−</i> spermatocytes at pachytene.

<p>Surface-spread meiotic chromosomes were coimmunostained with FK2 antibody (red) which recognizes both monoubiquitin and polyubiquitin conjugates and an antibody to SCP3 (green), a component of the synaptonemal complex. (<b>A</b>, <b>B</b>) Leptotene. (<b>C</b>, <b>D</b>) Zygotene. (<b>E</b>, <b>F</b>) Early pachytene. (<b>G</b>, <b>H</b>) Mid-pachytene. (<b>I</b>, <b>J</b>) Late pachytene. Compared with controls, the FK2 staining in <i>UBR2^−/−</i> chromosomes is relatively weak in the XY body at mid-pachytene and throughout the entire chromosomal regions at mid-late pachytene. Scale bar: 10 µm.</p

UBR2 is associated with chromatin during cell cycle of somatic cells.

<p>(<b>A</b>) UBR2 is enriched in the nucleus of MEFs. MEFs were stained for UBR2 without (top) and with a peptide that has been used to raise the antibody. (<b>B</b>) UBR2 is associated with chromatin in MEFs. Control and <i>UBR2^−/−</i> cells were separated into cytosolic, nuclear soluble, and chromatin-bound fractions in the presence or absence of iodoacetamide, followed by immunoblotting for proteins indicated. (<b>C</b>) Chromatin association of UBR2 is cell cycle-dependent. HeLa cells were synchronized at the G1-S border using the double thymidine block, released from G1-S arrest, and subjected to time-course fractionation and immunoblotting. Cell cycle stages were verified using flow cytometry and based on behaviors of cell cycle regulators, including down-regulation of chromatin-associated cyclin A and CDC6.</p

Chromosome instability and hypersensitivity to DNA damage of UBR2-deficient somatic cells.

<p>(<b>A</b>) UBR2-knockdown induces hyperproliferation in HeLa cells. (<b>B</b>) Metaphase chromosomes of <i>UBR2^−/−</i> MEFs show increased chromosomal aberrations, including breaks and fragmentations, compared with control cells. Arrowhead, break; Arrow, fragmentation (<b>C</b>) Quantitation of chromosomal abnormalities (breaks and fragments) observed in metaphase chromosomes from +/+ and <i>UBR2^−/−</i> MEFs. (<b>D</b>) <i>UBR2^−/−</i> MEFs are hypersensitive to hydroxyurea, or methyl methanesulfonate (Sigma).</p

Neonatal lethality in <i>UBR2^−/−</i> newborn pups associated with defects in lung expansion and neural development.

<p>(<b>A</b>) The majority of <i>UBR2^−/−</i> mice in the C57 genetic background die neonatally. Shown is gross morphology of neonatal pups enriched in the C57 genetic background at P1. Surviving <i>UBR2^−/−</i> neonates weighed slightly less than their +/+ and <i>UBR2^+/−</i> littermates of the same gender. No gross morphological differences were observed between <i>UBR2^−/−</i> and control mice during embryogenesis and after birth. Arrowheads mark stomachs with or without milk, which indicate the feeding from mother. (<b>B</b>) The lungs of <i>UBR2^−/−</i> newborn pups are not properly expanded. Shown are H&E-stained cross sections from +/+ and <i>UBR2^−/−</i> lungs at P0. Arrowheads indicate alveoli. (<b>C</b>) <i>In situ</i> hybridization of <i>UBR2</i> mRNA on cross sections of +/+ and <i>UBR2^−/−</i> brains at P1. (<b>D</b>) Dilated ventricles in <i>UBR2^−/−</i> brain (arrowhead). Shown are Nissl-stained cross sections of +/+ and <i>UBR2^−/−</i> brains at P1. (<b>E</b>) Defective gliogenesis and neuronal differentiation in hippocampus of <i>UBR2^−/−</i> brains at P1. Cross sections of a mildly affected <i>UBR2^−/−</i> brain, together with its littermate control, were subjected to Nissl or DAPI staining, or immunofluorescent staining of GFAP or NeuN.</p

Pachytene arrest of <i>UBR2^−/−</i> spermatocytes at stage IV.

<p>(<b>A</b>–<b>D</b>) Testis sections from 8-week (A–C) and 3-week (D) old <i>UBR2^−/−</i> tubules. (<b>A</b>) Tubules that did not yet reach epithelial stage IV. A large number of spermatocytes (thick arrow) are present, indicating that the spermatogonial compartment keeps forming spermatocytes. Arrowhead, diplotene spermatocyte; thin arrow, round spermatid. The predecessors of these cells survived the stage IV arrest. (<b>B</b>) Tubules in epithelial stage IV as evidenced by the presence of large, G2 phase intermediate (In) spermatogonia (blue arrow) about to or dividing into B spermatogonia (yellow arrowhead). There is massive apoptosis of spermatocytes (asterisks). (<b>C</b>) Tubules after stage IV. A variable number of spermatocytes survive the passage through stage IV. The left tubule shows only one spermatocyte (arrowhead) and a few round spermatids (thin arrow) that stem from spermatocytes that survived stage IV one epithelial cycle earlier. The tubule on the right shows more spermatocytes (arrowhead) and round spermatids (black arrow) and even a few elongated spermatids (yellow arrow). (<b>D</b>) Stage IV arrest at the age of 3 weeks. The lower tubule shows massive apoptosis (asterisk). The upper tubule is after stage IV and shows two surviving spermatocytes (arrowhead), indicating that the arrest was already present before three weeks. (<b>E</b>) Surface-spread chromosomes of 781 control and 691 <i>UBR2^−/−</i> spermatocytes isolated from mice at P17 were stained with SCP3 and staged based on the morphology of SCP3-positive chromosomes. (<b>F</b>) Surface-spread chromosomes of 344 +/+ and 161 <i>UBR2^−/−</i> pachytene spermatocytes were substaged.</p

UBR2 mediates monoubiquitylation and polyubiquitylation of H2A and H2B but not H3 and H4.

<p>(<b>A</b>) <i>In vitro</i> ubiquitylation assay (20 µL) with 1 µg of histone H2A, H2B, H1, or H3. The reaction contains 100 ng E3-F (or E3-V) prepared from rat testes, 30 ng UbcH2, and Ub activating reagents, including 1 µg flag-Ub and 100 ng E1. E3-F and E3-V represent protein mixtures that have been captured by Phe-peptide and Val-peptide, respectively. (<b>B</b>) <i>In vitro</i> binding assay in which UBR2 (as a mixture with UBR1) immobilized on Phe-peptide-beads was mixed with histone H2A, H2B, or H3 in the presence of HR6B, E1, and Ub activating reagents, followed by immunoblotting of histones retained by X-peptide (X = Phe or Val). (<b>C</b>) The screening of E2s which can support E3-F-mediated ubiquitylation of H2A. <i>In vitro</i> ubiquitylation assays were performed as (A) with different E2s indicated above. In this screening, UbcH2 and UbcH5b showed reproducibly the E2 activity in H2A ubiquitylation. (<b>D</b>–<b>F</b>) Allosteric modulation, an additional E2, and synthetic ligands for UBR2. (<b>D</b>) The interaction between UBR2 and HR6B is cooperatively accelerated by H2A and Arg-Ala. UBR2 (0.2 µg) from 10 mg rat testes extracts were immobilized with Phe-peptide conjugated with beads. Precipitated E3-peptide beads were mixed with 60 ng HR6B, 1 µg H2A, and/or 2 mM Arg-Ala, followed by immunoblotting analysis. (<b>E</b>) The HR6B-H2A interaction is cooperatively facilitated by UBR2 and Arg-Ala. GST-pulldown assays were done with 200 ng GST-HR6B, 200 ng UBR2, 1 µg H2A, and/or 2 mM Arg-Ala. (<b>F</b>) UBR2-dependent H2A ubiquitylation is synergistically activated by type-1 and type-2 N-end rule ligands.</p