The C-terminal domain is sufficient for host-binding activity of the Mu phage tail-spike protein

The Mu phage virion contains tail-spike proteins beneath the baseplate, which it uses to adsorb to the outer membrane of Escherichia coli during the infection process. The tail spikes are composed of gene product 45 (gp45), which contains 197 amino acid residues. In this study, we purified and characterized both the full-length and the C-terminal domains of recombinant gp45 to identify the functional and structural domains. Limited proteolysis resulted in a Ser64-Gln197 sequence, which was composed of a stable C-terminal domain. Analytical ultracentrifugation of the recombinant C-terminal domain (gp45-C) indicated that the molecular weight of gp45-C was about 58 kDa and formed a trimeric protomer in solution. Coprecipitation experiments and a quartz crystal microbalance (QCM) demonstrated that gp45-C irreversibly binds to the E. coli membrane. These results indicate that gp45 shows behaviors similar to tail-spike proteins of other phages; however, gp45 did not show significant sequence homology with the other phage tail-spike structures that have been identified.     

Distinct mechanisms of receptor and nonreceptor tyrosine kinase activation by reactive oxygen species in vascular smooth muscle cells: role of metalloprotease and protein kinase C-delta

Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor tyrosine kinase activation by H2O2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-delta activation, respectively. H2O2-induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H2O2-induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H2O2-induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H2O2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-delta transfection inhibited H2O2-induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H2O2-induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-delta inhibited PYK2 activation by H2O2. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-delta/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H2O2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.     

Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain-truncated form (HBdeltatm) of the molecule. HB(uc/uc) mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBdeltatm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.     

Differential expression of two cathepsin Es during metamorphosis-associated remodeling of the larval to adult type epithelium in Xenopus stomach

Cathepsin E (CE) was purified from the foregut of Xenopus laevis tadpoles as a mature dimeric form. The purified enzyme was a typical CE among aspartic proteinases with respect to pH dependence of proteolytic activity, susceptibility to pepstatin, and having N-linked high-mannose type oligosaccharide chains. We isolated two cDNAs for the CE (CE1 and CE2) from adult stomach. The amino acid sequence of the N-terminal region of the purified CE coincided with the corresponding sequence predicted from CE1. Northern blot analysis and in situ hybridization were performed. The CE1 mRNA was highly expressed in surface mucous cells and gland cells constituting the larval epithelium of the foregut of pro-metamorphic tadpoles. As metamorphosis began and progressed, CE1 mRNA drastically decreased in amount, and subsequently both CE1 and CE2 mRNAs gradually increased. The increase in CE2 mRNA was detected shortly after the increase in CE1 mRNA. The decrease in CE1 expression correlated with degeneration of the larval type epithelium, while the increases in both CE1 and CE2 expression correlated with formation of the adult type epithelium. Thus, cathepsin E gene expression was differentially regulated during metamorphosis-associated remodeling of the larval to adult type epithelium in stomach.     

Evidence for a phosphorylation-independent role for Ser 32 and 36 in proteasome inhibitor-resistant (PIR) IkappaBalpha degradation in B cells

Constitutive NF-kappaB activity has emerged as an important cell survival regulator. Canonical inducible NF-kappaB activation involves IkappaB kinase (IKK)-dependent dual phosphorylation of Ser 32 and 36 of IkappaBalpha to cause its beta-TrCP-dependent ubiquitylation and proteasomal degradation. We recently reported that constitutive NF-kappaB (p50/c-Rel) activity in WEHI231 B cells is maintained through proteasome inhibitor-resistant (PIR) IkappaBalpha degradation in a manner that requires Ser 32 and 36, without the requirement of a direct interaction with beta-TrCP. Here we specifically examined whether dual phosphorylation of Ser 32 and 36 was required for PIR degradation. Through mutagenesis studies, we found that dual replacement of Ser 32 and 36 with Glu permitted beta-TrCP and proteasome-dependent, but not PIR, degradation. Moreover, single replacement of either Ser residue with Leu permitted PIR degradation in WEHI231 B cells. These results indicate that PIR degradation occurs in the absence of dual phosphorylation, thereby explaining the beta-TrCP-independent nature of the PIR pathway. Additionally, we found evidence that PIR IkappaBalpha degradation controls constitutive NF-kappaB activation in certain multiple myeloma cells. These results suggest that B lineage cells can differentiate between PIR and canonical IkappaBalpha degradation through the absence or presence of dually phosphorylated IkappaBalpha.     

The HNF-1 target collectrin controls insulin exocytosis by SNARE complex formation

Defective glucose-stimulated insulin secretion is the main cause of hyperglycemia in type 2 diabetes mellitus. Mutations in HNF-1 cause a monogenic form of type 2 diabetes, maturity-onset diabetes of the young (MODY), characterized by impaired insulin secretion. Here we report that collectrin, a recently cloned kidney-specific gene of unknown function, is a target of HNF-1 in pancreatic β cells. Expression of collectrin was decreased in the islets of HNF-1 (−/−) mice, but was increased in obese hyperglycemic mice. Overexpression of collectrin in rat insulinoma INS-1 cells or in the β cells of transgenic mice enhanced glucose-stimulated insulin exocytosis, without affecting Ca²⁺ influx. Conversely, suppression of collectrin attenuated insulin secretion. Collectrin bound to SNARE complexes by interacting with snapin, a SNAP-25 binding protein, and facilitated SNARE complex formation. Therefore, collectrin is a regulator of SNARE complex function, which thereby controls insulin exocytosis.     

Cdc6 protein activates p27KIP1-bound Cdk2 protein only after the bound p27 protein undergoes C-terminal phosphorylation

In mammalian cells Cdk2 activity during the G(1)-S transition is mainly controlled by p27(KIP1). Although the amount and subcellular localization of p27 influence Cdk2 activity, how Cdk2 activity is regulated during this phase transition still remains virtually unknown. Here we report an entirely new mechanism for this regulation. Cdc6 the AAA+ ATPase, known to assemble prereplicative complexes on chromosomal replication origins and activate p21(CIP1)-bound Cdk2, also activated p27-bound Cdk2 in its ATPase and cyclin binding motif-dependent manner but only after the p27 bound to the Cdk2 was phosphorylated at the C terminus. ROCK, which mediates a signal for cell anchorage to the extracellular matrix and activates the mTORC1 cascade as well as controls cytoskeleton assembly, was partly responsible for C-terminal phosphorylation of the p27. In vitro reconstitution demonstrated ROCK (Rho-associated kinase)-mediated phosphorylation of Cdk2-bound p27 at the C terminus and subsequent activation of the Cdk2 by Cdc6.     

Identification of novel peptide agonists from a random peptide library for a 5-oxo-ETE receptor, a receptor for bioactive lipids

A combinatorial peptide library contains an enormous combination of amino acid sequences and drug candidates, but an effective screening strategy to identify a variety of bioactive peptides has yet to be established. In this article, a random hexapeptide library was screened to identify novel peptide ligands for a 5-oxo-ETE receptor (OXER), which is a G-protein-coupled receptor for bioactive lipids, by using an OXER-Gi1alpha fusion protein. We successfully identified 2 hexapeptides-Ac-HMQLYF-NH2 and Ac-HMWLYF-NH(2)-that exhibited agonistic activity. Although the corresponding affinities were relatively low (EC50 values of 146 and 6.7 microM, respectively), the activities were confirmed by other independent cell-based assay methods, namely, intracellular calcium mobilization and cell chemotaxis. This study demonstrates that a combinatorial peptide library may be screened using a [35S]GTPgammaS binding assay with G-protein-coupled receptor (GPCR)-Galpha fusion proteins, in general, and that of peptide ligands can be obtained even for nonpeptide receptors.     

An 83-kDa embryonic-type nuclear antigen is detected within the germinal vesicles of oocytes of the ascidianHalocynthia roretzi

Summary Monoclonal antibodies were raised against germinal vesicles which were isolated from fully grown oocytes of the ascidianHalocynthia roretzi. Immunoblot analyses revealed that one of the antibodies, designated Hgv-2, recognized a single band with a molecular weight of about 83 kDa. The antibody, visualized by indirect immunohistochemistry, reacted only with the germinal vesicles of oocytes and did not react with test cells, follicle cells, and other somatic cells of the gonad. During embryogenesis the antigenicity was found in interphase nuclei of all embryonic cells. The antibody did not react with chromosomes or the mitotic apparatus. The antigenicity was retained by interphase nuclei of larval cells, but it disappeared from nuclei of juveniles about 7 days after metamorphosis.     

Glycomics of a novel type-2 N-acetyllactosamine-specific lectin purified from the feather star, Oxycomanthus japonicus (Pelmatozoa: Crinoidea)

A lectin - designated OXYL for the purposes of this study that strongly recognizes complex-type oligosaccharides of serum glycoproteins - was purified from a crinoid, the feather star Oxycomanthus japonicus, the most basal group among extant echinoderms. OXYL was purified through a combination of anion-exchange and affinity chromatography using Q-sepharose and fetuin-sepharose gel, respectively. Lectin was determined to be a 14-kDa polypeptide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions. However, 14-kDa and 28-kDa bands appeared in the same proportion under non-reducing conditions. Gel permeation chromatography showed a 54-kDa peak, suggesting that lectin consists of four 14-kDa subunits. Divalent cations were not indicated, and stable haemagglutination activity was demonstrated at pH 4-12 and temperatures below 60°C. Surface plasmon resonance analysis of OXYL against fetuin showed k(ass) and k(diss) values of 1.4×10(-6)M(-1)s(-1) and 3.1×10(-3)s(-1), respectively, indicating that it has a strong binding affinity to the glycoprotein as lectin. Frontal affinity chromatography using 25 types of prydylamine-conjugated glycans indicated that OXYL specifically recognizes multi-antennary complex-type oligosaccharides containing type-2 N-acetyllactosamines (Galβ1-4GlcNAc) if α2-3-linked sialic acid is linked at the non-reducing terminal. However, type-1 N-acetyllactosamine (Galβ1-3GlcNAc) chains and α2-6-linked sialic acids were never recognized by OXYL. This profiling study showed that OXYL essentially recognizes β1-4-linkage at C-1 position and free OH group at C-6 position of Gal in addition to the conservation of N-acetyl groups at C-2 position and free OH groups at C-3 position of GlcNAc in N-acetyllactosamine. This is the first report on glycomics on a lectin purified from an echinoderm belonging to the subphylum Pelmatozoa.