The catalytic and lectin domains of UDP-GalNAc:polypeptide alpha-N-Acetylgalactosaminyltransferase function in concert to direct glycosylation site selection

UDP-GalNAc:polypeptide alpha-N-Acetylgalactosaminyltransferases (ppGalNAcTs), a family (EC 2.4.1.41) of enzymes that initiate mucin-type O-glycosylation, are structurally composed of a catalytic domain and a lectin domain. Previous studies have suggested that the lectin domain modulates the glycosylation of glycopeptide substrates and may underlie the strict glycopeptide specificity of some isoforms (ppGalNAcT-7 and -10). Using a set of synthetic peptides and glycopeptides based upon the sequence of the mucin, MUC5AC, we have examined the activity and glycosylation site preference of lectin domain deletion and exchange constructs of the peptide/glycopeptide transferase ppGalNAcT-2 (hT2) and the glycopeptide transferase ppGalNAcT-10 (hT10). We demonstrate that the lectin domain of hT2 directs glycosylation site selection for glycopeptide substrates. Pre-steady-state kinetic measurements show that this effect is attributable to two mechanisms, either lectin domain-aided substrate binding or lectin domain-aided product release following glycosylation. We find that glycosylation of peptide substrates by hT10 requires binding of existing GalNAcs on the substrate to either its catalytic or lectin domain, thereby resulting in its apparent strict glycopeptide specificity. These results highlight the existence of two modes of site selection used by these ppGalNAcTs: local sequence recognition by the catalytic domain and the concerted recognition of distal sites of prior glycosylation together with local sequence binding mediated, respectively, by the lectin and catalytic domains. The latter mode may facilitate the glycosylation of serine or threonine residues, which occur in sequence contexts that would not be efficiently glycosylated by the catalytic domain alone. Local sequence recognition by the catalytic domain differs between hT2 and hT10 in that hT10 requires a pre-existing GalNAc residue while hT2 does not.     

Rtf1-mediated eukaryotic site-specific replication termination

The molecular mechanisms mediating eukaryotic replication termination and pausing remain largely unknown. Here we present the molecular characterization of Rtf1 that mediates site-specific replication termination at the polar Schizosaccharomyces pombe barrier RTS1. We show that Rtf1 possesses two chimeric myb/SANT domains: one is able to interact with the repeated motifs encoded by the RTS1 element as well as the elements enhancer region, while the other shows only a weak DNA binding activity. In addition we show that the C-terminal tail of Rtf1 mediates self-interaction, and deletion of this tail has a dominant phenotype. Finally, we identify a point mutation in Rtf1 domain I that converts the RTS1 element into a replication barrier of the opposite polarity. Together our data establish that multiple protein DNA and protein-protein interactions between Rtf1 molecules and both the repeated motifs and the enhancer region of RTS1 are required for site-specific termination at the RTS1 element.     

Multiple roles for nuclear localization signal (NLS, aa 442-472) of receptor interacting protein 3 (RIP3)

RIP3, a Ser/Thr kinase of RIP (Receptor Interacting Protein) family, is recruited to the TNFR1 signaling complex through RIP and has been shown to mediate apoptosis induction and NF-κB activation. RIP3 is a nucleocytoplasmic shuttling protein and its unconventional nuclear localization signal (NLS, 442-472 aa) is sufficient to trigger apoptosis in the nucleus. In this study, we demonstrate that this NLS exhibits several other roles besides apoptotic function. Firstly, this NLS was found to be required for both RIP3-induced apoptosis and RIP3-mediated NF-κB activation. Next, similar to RHIM motif (RIP homotypic interaction motif), NLS of RIP3 was found to be involved in RIP3-RIP interaction. Furthermore, this NLS was found to be both sufficient and necessary for RIP3 self-association. Our primary data also showed that RIP3 might form a homodimer within cells, and its apoptotic activity may not be required for this dimerization, rather the intactness of NLS determines RIP3-induced apoptosis, since a point mutation at amino acid residue 452 (Ile to Ala) within NLS greatly reduced its apoptotic ability, despite that RIP3 point mutant RIP3/I452A is able to dimerize with wild type RIP3 or itself.     

秉螈Pingia granulosus的重新发现及新模描述

1933年,张孟闻以1号采自浙江天台山的次成体有尾类标本定名粗皮肥螈Pachytriton granulosus,并于1935年建立新属--秉螈属Pingia以辖之.因模式标本在日本侵华战争期间遗失且多年来一直未再采集到标本,后来学者多认为其是无斑肥螈的同物异名.2008年6月1日,两栖动物爱好者周行在浙江龙王山采集到2号有尾类标本,经本文作者仔细研究后发现,除部分色斑及指(趾)端腹面未覆有角质鞘外,其他特征与秉螈Pingia granu.losus完全相符.因此作者建议恢复秉螈属Pingia和秉螈Pingia granulosus的有效性,并指定新模标本予以描述.     

GAK is phosphorylated by c-Src and translocated from the centrosome to chromatin at the end of telophase

Cyclin G-associated kinase (GAK) harbors a consensus phosphorylation motif (Y412) for c-Src; however, its physiological significance remains elusive. Here, we show that GAK is phosphorylated by c-Src not only at Y412 but also at Y1149. An anti-GAK-pY412 antibody recognized the shifted band of GAK during M phase. Immunofluorescence (IF) showed that GAK-pY412/pY1149 signals were present in the nucleus during interphase, translocated to chromosomes at prophase and prometaphase, moved to centrosomes at metaphase, and finally translocated to chromosomes at the end of telophase, when nuclear membrane formation was almost complete. These subcellular movements of GAK resemble those of DNA licensing factors. Indeed, mass spectrometry identified mini-chromosome maintenance (MCM) 3, an essential component of the DNA licensing system, as one of the association partners of GAK; immunoprecipitation-mediated Western blotting confirmed their association in vivo. These results suggest that the c-Src_GAK_MCM axis plays an important role in cell cycle progression through control of the DNA replication licensing system.     

Exploratory differential gene expression analysis in microarray experiments with no or limited replication

We describe an exploratory, data-oriented approach for identifying candidates for differential gene expression in cDNA microarray experiments in terms of α-outliers and outlier regions, using simultaneous tolerance intervals relative to the line of equivalence (Cy5 = Cy3). We demonstrate the improved performance of our approach over existing single-slide methods using public datasets and simulation studies.     

mRNA degradation by the virion host shutoff (Vhs) protein of herpes simplex virus: genetic and biochemical evidence that Vhs is a nuclease

During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating the decay of all mRNAs, it helps redirect the cell from host to viral gene expression and facilitates the sequential expression of different classes of viral genes. While it is clear that Vhs induces mRNA degradation, it is uncertain whether it is itself an RNase or somehow activates a cellular enzyme. This question was addressed by using a combination of genetic and biochemical approaches. The Vhs homologues of alphaherpesviruses share sequence similarities with a family of mammalian, yeast, bacterial, and phage nucleases. To test the functional significance of these similarities, Vhs was mutated to alter residues corresponding to amino acids known to be critical to the nuclease activity of cellular homologues. In every instance, mutations that inactivated the nuclease activity of cellular homologues also abolished Vhs activity. Recent experiments showed that Vhs interacts with the cellular translation initiation factor eIF4H. In this study, the coexpression of Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein in bacteria resulted in the formation of a complex of the proteins. The wild-type Vhs/GST-eIF4H complex was isolated and shown to have RNase activity. In contrast, Vhs mutations that altered key residues in the nuclease motif abolished the nuclease activity of the recombinant Vhs/GST-eIF4H complex. The results provide genetic and biochemical evidence that Vhs is an RNase, either alone or as a complex with eIF4H.