The catalytically inactive tyrosine phosphatase HD-PTP/PTPN23 is a novel regulator of SMN complex localization

The survival motor neuron (SMN) complex fulfils essential functions in the assembly of snRNPs, which are key components in the splicing of pre-mRNAs. Little is known about the regulation of SMN complex activity by posttranslational modification despite its complicated phosphorylation pattern. Several phosphatases had been implicated in the regulation of SMN, including the nuclear phosphatases PPM1G and PP1γ. Here we systematically screened all human phosphatase gene products for a regulatory role in the SMN complex. We used the accumulation of SMN in Cajal bodies of intact proliferating cells, which actively assemble snRNPs, as a readout for unperturbed SMN complex function. Knockdown of 29 protein phosphatases interfered with SMN accumulation in Cajal bodies, suggesting impaired SMN complex function, among those the catalytically inactive, non–receptor-type tyrosine phosphatase PTPN23/HD-PTP. Knockdown of PTPN23 also led to changes in the phosphorylation pattern of SMN without affecting the assembly of the SMN complex. We further show interaction between SMN and PTPN23 and document that PTPN23, like SMN, shuttles between nucleus and cytoplasm. Our data provide the first comprehensive screen for SMN complex regulators and establish a novel regulatory function of PTPN23 in maintaining a highly phosphorylated state of SMN, which is important for its proper function in snRNP assembly.     

Generation and characterization of ABT-981, a dual variable domain immunoglobulin (DVD-IgTM) molecule that specifically and potently neutralizes both IL-1α and IL-1β

Interleukin-1 (IL-1) cytokines such as IL-1α, IL-1β, and IL-1Ra contribute to immune regulation and inflammatory processes by exerting a wide range of cellular responses, including expression of cytokines and chemokines, matrix metalloproteinases, and nitric oxide synthetase. IL-1α and IL-1β bind to IL-1R1 complexed to the IL-1 receptor accessory protein and induce similar physiological effects. Preclinical and clinical studies provide significant evidence for the role of IL-1 in the pathogenesis of osteoarthritis (OA), including cartilage degradation, bone sclerosis, and synovial proliferation. Here, we describe the generation and characterization of ABT-981, a dual variable domain immunoglobulin (DVD-Ig) of the IgG1/k subtype that specifically and potently neutralizes IL-1α and IL-1β. In ABT-981, the IL-1β variable domain resides in the outer domain of the DVD-Ig, whereas the IL-1α variable domain is located in the inner position. ABT-981 specifically binds to IL-1α and IL-1β, and is physically capable of binding 2 human IL-1α and 2 human IL-1β molecules simultaneously. Single-dose intravenous and subcutaneous pharmacokinetics studies indicate that ABT-981 has a half-life of 8.0 to 10.4 d in cynomolgus monkey and 10.0 to 20.3 d in rodents. ABT-981 exhibits suitable drug-like-properties including affinity, potency, specificity, half-life, and stability for evaluation in human clinical trials. ABT-981 offers an exciting new approach for the treatment of OA, potentially addressing both disease modification and symptom relief as a disease-modifying OA drug.     

Polysialic Acid on Neuropilin-2 Is Exclusively Synthesized by the Polysialyltransferase ST8SiaIV and Attached to Mucin-type O-Glycans Located between the b2 and c Domain

Neuropilin-2 (NRP2) is well known as a co-receptor for class 3 semaphorins and vascular endothelial growth factors, involved in axon guidance and angiogenesis. Moreover, NRP2 was shown to promote chemotactic migration of human monocyte-derived dendritic cells (DCs) toward the chemokine CCL21, a function that relies on the presence of polysialic acid (polySia). In vertebrates, this posttranslational modification is predominantly found on the neural cell adhesion molecule (NCAM), where it is synthesized on N-glycans by either of the two polysialyltransferases, ST8SiaII or ST8SiaIV. In contrast to NCAM, little is known on the biosynthesis of polySia on NRP2. Here we identified the polySia attachment sites and demonstrate that NRP2 is recognized only by ST8SiaIV. Although polySia-NRP2 was found on bone marrow-derived DCs from wild-type and St8sia2^−/− mice, polySia was completely lost in DCs from St8sia4^−/− mice despite normal NRP2 expression. In COS-7 cells, co-expression of NRP2 with ST8SiaIV but not ST8SiaII resulted in the formation of polySia-NRP2, highlighting distinct acceptor specificities of the two polysialyltransferases. Notably, ST8SiaIV synthesized polySia selectively on a NRP2 glycoform that was characterized by the presence of sialylated core 1 and core 2 O-glycans. Based on a comprehensive site-directed mutagenesis study, we localized the polySia attachment sites to an O-glycan cluster located in the linker region between b2 and c domain. Combined alanine exchange of Thr-607, -613, -614, -615, -619, and -624 efficiently blocked polysialylation. Restoration of single sites only partially rescued polysialylation, suggesting that within this cluster, polySia is attached to more than one site.     

Deciphering the assembly pathway of Sm-class U snRNPs

The assembly of the Sm-class of uridine-rich small nuclear ribonucleoproteins (U snRNPs), albeit spontaneous in vitro, has recently been shown to be dependent on the aid of a large number of assisting factors in vivo. These factors are organized in two interacting units termed survival motor neuron (SMN)- and protein arginine methyltransferase 5 (PRMT5)-complexes, respectively. While the PRMT5-complex acts early in the assembly pathway by activating common proteins of U snRNPs, the SMN-complex functions to join proteins and RNA in a highly ordered, apparently regulated manner. Here, we summarize recent progress in the understanding of this process and discuss the influence exerted by the aforementioned trans-acting factors.     

Ikaros regulates Notch target gene expression in developing thymocytes

Both Ikaros and Notch are essential for normal T cell development. Collaborative mutations causing a reduction in Ikaros activity and an increase in Notch activation promote T cell leukemogenesis. Although the molecular mechanisms of this cooperation have been studied, its consequences in thymocyte development remain unexplored. In this study, we show that Ikaros regulates expression of a subset of Notch target genes, including Hes1, Deltex1, pTa, Gata3, and Runx1, in both Ikaros null T cell leukemia lines and Ikaros null primary thymocytes. In Ikaros null leukemia cells, Notch deregulation occurs at both the level of Notch receptor cleavage and expression of Notch target genes, because re-expression of Ikaros in these cells down-regulates Notch target gene expression without affecting levels of intracellular cleaved Notch. In addition, abnormal expression of Notch target genes is observed in Ikaros null double-positive thymocytes, in the absence of detectable intracellular cleaved Notch. Finally, we show that this role of Ikaros is specific to double-positive and single-positive thymocytes because derepression of Notch target gene expression is not observed in Ikaros null double-negative thymocytes or lineage-depleted bone marrow. Thus, in this study, we provide evidence that Ikaros and Notch play opposing roles in regulation of a subset of Notch target genes and that this role is restricted to developing thymocytes where Ikaros is required to appropriately regulate the Notch program as they progress through T cell development.     

Retention of plastic-tipped dart tags in African tigerfish Hydrocynus vittatus

Estimates of tag retention and tagging-related mortality are essential for mark-recapture experiments. Mortality and tag loss were estimated from 15 tigerfish Hydrocynus vittatus marked using Hallmark model PDL plastic-tipped dart tags released into a 1 730 m² pond at Kamutjonga Inland Fisheries Institute, Namibia, and inspected bi-monthly for the presence or absence of tags. No mortality was observed during the experiment. All marked fish had lost their tags after 10 months and 50% tag loss was estimated at 3.9 months. The high tag loss rate indicates that PDL plastic-tipped dart tags are not suitable for long-term studies on this species.     

Synthesis of potent taxoids for tumor-specific delivery using monoclonal antibodies

The targeted delivery of taxoids, in the form of taxane-antibody immunoconjugates, requires the preparation of taxoids containing moieties suitable for their conjugation to monoclonal antibodies. A series of taxoids incorporating a disulfide-containing linker at various positions of the taxoid framework have been prepared to investigate the most suitable position for conjugation. A second series of taxoids modified at the C-2 position aimed at increasing the potency of these taxanes has also been prepared.     

Fos in the suprachiasmatic nucleus of house mouse lines that reveal a different phase-delay response to the same light pulse

Increased light intensity of a 5-min light pulse is positively correlated with Fos mRNA and Fos protein levels in the suprachiasmatic nucleus (SCN) of hamsters. These findings suggest that the level of Fos activation is proportional to the light intensity and that the magnitude of the phase-shift response depends on the level of Fos activation. However, to what extent different phase-delay responses to the same light pulse are mediated by differential Fos activation is unknown. To elucidate this, the authors used selected house mouse lines that reveal an almost threefold difference in phase-delay responses in constant darkness (DD) between circadian time (CT) 16 and CT 20 to the same light pulse. The authors measured wheel-running activity and subjected male mice of these lines to a 15-min light pulse at CT 16 after 2 weeks in DD. The behavioral response was measured and 10 to 12 days later the animals were again subjected to the same light pulse at CT 16. One hour after the start of the second light pulse, the animals were sacrificed for Fos immunocytochemistry. Results indicate a significant difference between the lines in the phase-delay response (F2,26 = 5.112, p < 0.017) and the level of Fos activation (F2,26 = 27.15, p < 0.0001) after a 15-min light pulse at CT 16. These findings support the hypothesis that the magnitude of the phase-delay response is proportional to the number of cells in the SCN that exhibit Fos induction after the same 15-min light pulse at CT 16 in DD. It also indicates a possible difference in the input pathways among the lines.