43 research outputs found

    Activation of ERK and NF-κB during HARE-Mediated Heparin Uptake Require Only One of the Four Endocytic Motifs

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    We thank Bruce A. Baggenstoss and Jennifer L. Washburn for technical support in many experiments and Emma K. Blank, Andrew W. Egger, Brianna M. Kellar and Helen T. Russom for assistance with experiments supporting Fig 2.Fifteen different ligands, including heparin (Hep), are cleared from lymph and blood by the Hyaluronan (HA) Receptor for Endocytosis (HARE; derived from Stabilin-2 by proteolysis), which contains four endocytic motifs (M1-M4). Endocytosis of HARE•Hep complexes is targeted to coated pits by M1, M2, and M3 (Pandey et al, Int. J. Cell Biol. 2015, article ID 524707), which activates ERK1/2 and NF-κB (Pandey et al J. Biol. Chem. 288, 14068–79, 2013). Here, we used a NF-κB promoter-driven luciferase gene assay and cell lines expressing different HARE cytoplasmic domain mutants to identify motifs needed for Hep-mediated signaling. Deletion of M1, M2 or M4 singly had no effect on Hep-mediated ERK1/2 activation, whereas signaling (but not uptake) was eliminated in HARE(ΔM3) cells lacking NPLY2519. ERK1/2 signaling in cells expressing WT HARE(Y2519A) or HARE(Y2519A) lacking M1, M2 and M4 (containing M3-only) was decreased by 75% or eliminated, respectively. Deletion of M3 (but not M1, M2 or M4) also inhibited the formation of HARE•Hep•ERK1/2 complexes by 67%. NF-κB activation by HARE-mediated uptake of Hep, HA, dermatan sulfate or acetylated LDL was unaffected in single-motif deletion mutants lacking M1, M2 or M4. In contrast, cells expressing HARE(ΔM3) showed loss of HARE-mediated NF-κB activation during uptake of each of these four ligands. NF-κB activation by the four signaling ligands was also eliminated in HARE(Y2519A) or HARE(M3-only;Y2519A) cells. We conclude that the HARE NPLY2519 motif is necessary for both ERK1/2 and NF-κB signaling and that Tyr2519 is critical for these functions.Yeshttp://www.plosone.org/static/editorial#pee

    Evaluation of MC1R high-throughput nucleotide sequencing data generated by the 1000 Genomes Project

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    Abstract The advent of next-generation sequencing allows simultaneous processing of several genomic regions/individuals, increasing the availability and accuracy of whole-genome data. However, these new approaches may present some errors and bias due to alignment, genotype calling, and imputation methods. Despite these flaws, data obtained by next-generation sequencing can be valuable for population and evolutionary studies of specific genes, such as genes related to how pigmentation evolved among populations, one of the main topics in human evolutionary biology. Melanocortin-1 receptor (MC1R) is one of the most studied genes involved in pigmentation variation. As MC1R has already been suggested to affect melanogenesis and increase risk of developing melanoma, it constitutes one of the best models to understand how natural selection acts on pigmentation. Here we employed a locally developed pipeline to obtain genotype and haplotype data for MC1R from the raw sequencing data provided by the 1000 Genomes FTP site. We also compared such genotype data to Phase 3 VCF to evaluate its quality and discover any polymorphic sites that may have been overlooked. In conclusion, either the VCF file or one of the presently described pipelines could be used to obtain reliable and accurate genotype calling from the 1000 Genomes Phase 3 data

    Sugars in the microenvironment: the sticky problem of HA turnover in tumors

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