New syntax to describe local continuous structure-sequence information for recognizing new pre-miRNAs

As an important complement to experimental identification of pre-miRNA, computational prediction methods are attracting more and more attention. Features extracted from pre-miRNA are the key to computational prediction. Among the features, local continuous structure-sequence information is usually employed by existing computational methods. As more and more species-specific miRNAs have been identified, a new syntax is required to describe pre-miRNA local continuous structure-sequence features. Therefore, we proposed here the use of couplet syntax to describe pre-miRNA intrinsic features. When tested on a dataset from miRBase12.0 with the use of features extracted by couplet syntax, the SVM classifier achieves a sensitivity of 81.98% and specificity of 87.16% on a human test set and a sensitivity of 86.71% on all other species. The obtained results indicate that the proposed couplet syntax can describe the intrinsic features of pre-miRNA better than traditional methods. By means of describing pre-miRNA secondary structure more precisely and masking frequently mutated nucleotides, couplet syntax provides a powerful feature-describing method that can be applied to many computational prediction methods.     

Enhancing Antibody Fc Heterodimer Formation through Electrostatic Steering Effects: APPLICATIONS TO BISPECIFIC MOLECULES AND MONOVALENT IgG

Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. However, co-expression of multiple light and heavy chains often leads to contaminants and pose purification challenges. In this work, we have modified the CH3 domain interface of the antibody Fc region with selected mutations so that the engineered Fc proteins preferentially form heterodimers. These novel mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation. This new Fc heterodimer format was used to produce bispecific single chain antibody fusions and monovalent IgGs with minimal homodimer contaminants. The strategy proposed here demonstrates the feasibility of robust production of novel Fc-based heterodimeric molecules and hence broadens the scope of bispecific molecules for therapeutic applications.     

Inhibitory Phosphorylation of GSK-3 by CaMKII Couples Depolarization to Neuronal Survival

Glycogen synthase kinase-3 (GSK-3) plays a critical role in neuronal apoptosis. The two mammalian isoforms of the kinase, GSK-3α and GSK-3β, are inhibited by phosphorylation at Ser-21 and Ser-9, respectively. Depolarization, which is vital for neuronal survival, causes both an increase in Ser-21/9 phosphorylation and an inhibition of GSK-3α/β. However, the role of GSK-3 phosphorylation in depolarization-dependent neuron survival and the signaling pathway contributing to GSK-3 phosphorylation during depolarization remain largely unknown. Using several approaches, we showed that both isoforms of GSK-3 are important for mediating neuronal apoptosis. Nonphosphorylatable GSK-3α/β mutants (S21A/S9A) promoted apoptosis, whereas a peptide encompassing Ser-9 of GSK-3β protected neurons in a phosphorylation-dependent manner; these results indicate a critical role for Ser-21/9 phosphorylation on depolarization-dependent neuron survival. We found that Ser-21/9 phosphorylation of GSK-3 was mediated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) but not by Akt/PKB, PKA, or p90^RSK. CaMKII associated with and phosphorylated GSK-3α/β. Furthermore, the pro-survival effect of CaMKII was mediated by GSK-3 phosphorylation and inactivation. These findings identify a novel Ca²⁺/calmodulin/CaMKII/GSK-3 pathway that couples depolarization to neuronal survival.     

Extracellular High Mobility Group Box-1 (HMGB1) Inhibits Enterocyte Migration via Activation of Toll-like Receptor-4 and Increased Cell-Matrix Adhesiveness

Toll-like receptor-4 (TLR4) is the receptor for bacterial lipopolysaccharide, yet it may also respond to a variety of endogenous molecules. Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in newborn infants and is characterized by intestinal mucosal destruction and impaired enterocyte migration due to increased TLR4 signaling on enterocytes. The endogenous ligands for TLR4 that lead to impaired enterocyte migration remain unknown. High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. We thus hypothesize that extracellular HMGB1 inhibits enterocyte migration via activation of TLR4 and sought to define the pathways involved. We now demonstrate that murine and human NEC are associated with increased intestinal HMGB1 expression, that serum HMGB1 is increased in murine NEC, and that HMGB1 inhibits enterocyte migration in vitro and in vivo in a TLR4-dependent manner. This finding was unique to enterocytes as HMGB1 enhanced migration of inflammatory cells in vitro and in vivo. In seeking to understand the mechanisms involved, TLR4-dependent HMGB1 signaling increased RhoA activation in enterocytes, increased phosphorylation of focal adhesion kinase, and increased phosphorylation of cofilin, resulting in increased stress fibers and focal adhesions. Using single cell force traction microscopy, the net effect of HMGB1 signaling was a TLR4-dependent increase in cell force adhesion, accounting for the impaired enterocyte migration. These findings demonstrate a novel pathway by which TLR4 activation by HMGB1 delays mucosal repair and suggest a novel potential therapeutic target in the amelioration of intestinal inflammatory diseases like NEC.     

Selective Translational Control of the Alzheimer Amyloid Precursor Protein Transcript by Iron Regulatory Protein-1

Iron influx increases the translation of the Alzheimer amyloid precursor protein (APP) via an iron-responsive element (IRE) RNA stem loop in its 5′-untranslated region. Equal modulated interaction of the iron regulatory proteins (IRP1 and IRP2) with canonical IREs controls iron-dependent translation of the ferritin subunits. However, our immunoprecipitation RT-PCR and RNA binding experiments demonstrated that IRP1, but not IRP2, selectively bound the APP IRE in human neural cells. This selective IRP1 interaction pattern was evident in human brain and blood tissue from normal and Alzheimer disease patients. We computer-predicted an optimal novel RNA stem loop structure for the human, rhesus monkey, and mouse APP IREs with reference to the canonical ferritin IREs but also the IREs encoded by erythroid heme biosynthetic aminolevulinate synthase and Hif-2α mRNAs, which preferentially bind IRP1. Selective 2′-hydroxyl acylation analyzed by primer extension analysis was consistent with a 13-base single-stranded terminal loop and a conserved GC-rich stem. Biotinylated RNA probes deleted of the conserved CAGA motif in the terminal loop did not bind to IRP1 relative to wild type probes and could no longer base pair to form a predicted AGA triloop. An AGU pseudo-triloop is key for IRP1 binding to the canonical ferritin IREs. RNA probes encoding the APP IRE stem loop exhibited the same high affinity binding to rhIRP1 as occurs for the H-ferritin IRE (35 pm). Intracellular iron chelation increased binding of IRP1 to the APP IRE, decreasing intracellular APP expression in SH-SY5Y cells. Functionally, shRNA knockdown of IRP1 caused increased expression of neural APP consistent with IRP1-APP IRE-driven translation.     

Functional Interaction of Phosphatase and Tensin Homologue (PTEN) with the E3 Ligase NEDD4-1 during Neuronal Response to Zinc

The contribution of zinc-mediated neuronal death in the process of both acute and chronic neurodegeneration has been increasingly appreciated. Phosphatase and tensin homologue, deleted on chromosome 10 (PTEN), the major tumor suppressor and key regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, plays a critical role in neuronal death in response to various insults. NEDD4-1-mediated PTEN ubiquitination and subsequent degradation via the ubiquitin proteosomal system have recently been demonstrated to be the important regulatory mechanism for PTEN in several cancer types. We now demonstrate that PTEN is also the key mediator of the PI3K/Akt pathway in the neuronal response to zinc insult. We used primary cortical neurons and neuroblastoma N2a cells to show that zinc treatment results in a reduction of the PTEN protein level in parallel with increased NEDD4-1 gene/protein expression. The reduced PTEN level is associated with an activated PI3K pathway as determined by elevated phosphorylation of both Akt and GSK-3 as well as by the attenuating effect of a specific PI3K inhibitor (wortmannin). The reduction of PTEN can be attributed to increased protein degradation via the ubiquitin proteosomal system, as we show NEDD4-1 to be the major E3 ligase responsible for PTEN ubiquitination in neurons. Moreover, PTEN and NEDD4-1 appear to be able to counter-regulate each other to mediate the neuronal response to zinc. This reciprocal regulation requires the PI3K signaling pathway, suggesting a feedback loop mechanism. This study demonstrates that NEDD4-1-mediated PTEN ubiquitination is crucial in the regulation of PI3K/Akt signaling by PTEN during the neuronal response to zinc, which may represent a common mechanism in neurodegeneration.     

Na/H Exchanger Regulatory Factors Control Parathyroid Hormone Receptor Signaling by Facilitating Differential Activation of Gα Protein Subunits

The Na/H exchanger regulatory factors, NHERF1 and NHERF2, are adapter proteins involved in targeting and assembly of protein complexes. The parathyroid hormone receptor (PTHR) interacts with both NHERF1 and NHERF2. The NHERF proteins toggle PTHR signaling from predominantly activation of adenylyl cyclase in the absence of NHERF to principally stimulation of phospholipase C when the NHERF proteins are expressed. We hypothesized that this signaling switch occurs at the level of the G protein. We measured G protein activation by [³⁵S]GTPγS binding and Gα subtype-specific immunoprecipitation using three different cellular models of PTHR signaling. These studies revealed that PTHR interactions with NHERF1 enhance receptor-mediated stimulation of Gα_q but have no effect on stimulation of Gα_i or Gα_s. In contrast, PTHR associations with NHERF2 enhance receptor-mediated stimulation of both Gα_q and Gα_i but decrease stimulation of Gα_s. Consistent with these functional data, NHERF2 formed cellular complexes with both Gα_q and Gα_i, whereas NHERF1 was found to interact only with Gα_q. These findings demonstrate that NHERF interactions regulate PTHR signaling at the level of G proteins and that NHERF1 and NHERF2 exhibit isotype-specific effects on G protein activation.     

Syntheses, crystal structures and magnetic properties of 1D and 2D cobaltous coordination polymers with mixed ligands

Two new Co(II) coordination polymers with mixed ligands, {[Co(BTA)_0.5(DBI)₂]·DBI·H₂O}_n (1) and [Co(PDA)(DBI)(H₂O)]_n (2) (H₄BTA = benzene-1,2,4,5-tetracarboxylic acid; H₂PDA = 2,2′-(1,2-phenylene)diacetic acid; DBI = 5,6-dimethyl-1H-benzoimidazole) have been synthesized under hydrothermal conditions, respectively. Both of them are characterized by elemental analyses, powder X-ray diffraction, thermogravimetric analysis, single-crystal X-ray diffraction, and magnetic susceptibilities. In 1, the Co(II) ions are four-coordinated and lie in distorted tetrahedron coordination environment. 1D ladder-like chain structure is formed by the bridging BTA⁴⁻ ligand. In 2, the Co(II) ions are in slightly distorted octahedral coordination geometry, and linked by PDA²⁻ ligand exhibiting a 2D layer structure. Temperature-dependent magnetic susceptibility measurements of 1 and 2 revealed that there are antiferromagnetic interactions between Co(II) ions.