32 research outputs found
B cell emigration directly from the cortex of lymphoid follicles in the bursa of Fabricius
No full textProperties of a disease-specific prion probe
No full textIn a recently published article, Paramithiotis et al. describe antibodies specific for the prion Tyr-Tyr-Arg (YYR) repeat motif. These antibodies interact with the pathological isoform of the prion protein (PrPSC), but not with the normal cellular isoform (PrpC). Because of this restricted specificity, they suggest that YYR-specific antibodies could be useful for the diagnosis and treatment of prion diseases (Fig. O. The monoclonal antibodies, all of the IgM isotype, were produced by immunizing mice with a synthetic peptide (CYYRRYYRYY). When coupled to magnetic beads, these YYR-specific antibodies immunoprecipitate Prpsc much more efficiently than PrpC. Notably, the Paramithiotis study did not rely on antibodies to YYR for specific detection of PrP. Their immunoblots were not ultimately probed with Prpsc-specific antibodies, but rather with \u27regular\u27 antibodies. The latter can detect PrP (but do not distinguish between Prpsc and PrpC) in a precipitate that could include any protein containing solvent-accessible tyrosine and arginine residues. This report is notably similar to that of Korth et al. 2, who described a Prpsc-specific IgM (designated 15B3) after immunizing with full-length recombinant bovine PrP. The 15B3 epitope consists of three separate, linear segments of PrP (15B3-1, 15B3-2 and 15B3-3). The YYR epitope (bold) identified by Paramithiotis et al. is included in or located near two of the 15B3 segments (underlined): GSDYEDRYYR (l5B3-1) and YYRPVDOYS (l5B3-2). Thus, these two independent studies relying on the same method of immunoprecipitation have identified similar IgM antibodies interacting with the same region on PrP, and possibly with the same YYR motifs
Proprotein convertase subtilisin/kexin type 9 (PCSK9) can mediate degradation of the low density lipoprotein receptor-related protein 1 (LRP-1).
No full textElevated LDL-cholesterol (LDLc) levels are a major risk factor for cardiovascular disease and atherosclerosis. LDLc is cleared from circulation by the LDL receptor (LDLR). Proprotein convertase subtilisin/kexin 9 (PCSK9) enhances the degradation of the LDLR in endosomes/lysosomes, resulting in increased circulating LDLc. PCSK9 can also mediate the degradation of LDLR lacking its cytosolic tail, suggesting the presence of as yet undefined lysosomal-targeting factor(s). Herein, we confirm this, and also eliminate a role for the transmembrane-domain of the LDLR in mediating its PCSK9-induced internalization and degradation. Recent findings from our laboratory also suggest a role for PCSK9 in enhancing tumor metastasis. We show herein that while the LDLR is insensitive to PCSK9 in murine B16F1 melanoma cells, PCSK9 is able to induce degradation of the low density lipoprotein receptor-related protein 1 (LRP-1), suggesting distinct targeting mechanisms for these receptors. Furthermore, PCSK9 is still capable of acting upon the LDLR in CHO 13-5-1 cells lacking LRP-1. Conversely, PCSK9 also acts on LRP-1 in the absence of the LDLR in CHO-A7 cells, where re-introduction of the LDLR leads to reduced PCSK9-mediated degradation of LRP-1. Thus, while PCSK9 is capable of inducing degradation of LRP-1, the latter is not an essential factor for LDLR regulation, but the LDLR effectively competes with LRP-1 for PCSK9 activity. Identification of PCSK9 targets should allow a better understanding of the consequences of PCSK9 inhibition for lowering LDLc and tumor metastasis
PCSK9 acts on the LDLR independent of the receptor's CT and TMD.
No full text<p><b>A</b>) Generation of chimeric truncated LDLR-V5 constructs. Schematic representation of the LDLR, LDLR lacking its CT (ΔCT), and ΔCT in which the LDLR TMD was swapped with that of ACE2 (ΔCT<sub>TMDace2</sub>) or VLDLR (ΔCT<sub>TMDvldlr</sub>). All constructs contained a C-terminal V5-tag. <b>B</b>) Expression in HEK293 cells. WT and chimeric LDLR constructs were transfected in HEK293 cells. Construct expression was assessed by immunoblotting with mAb-V5. Both mature and immature forms of the LDLR were detected. β-actin was used as a loading control. <b>C)</b> PCSK9 induces LDLR degradation independent of the LDLR's CT and TMD. LDLR, ΔCT, and the ΔCT<sub>TMDace2</sub> and ΔCT<sub>TMDvldlr</sub> chimeric constructs were expressed in HEK293 cells. Twenty-four hours post-transfection, the cells were treated overnight with empty vector control pIRES-V5 or PCSK9-V5 conditioned media, which contains both full length PCSK9 and its furin cleaved product at Arg<sub>218</sub>, PCSK9-ΔN<sub>218 </sub><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064145#pone.0064145-Benjannet4" target="_blank">[33]</a>. Cells were lysed in 1x RIPA and subjected to Western blot analysis. LDLR and PCSK9 were detected with mAb-V5. β-actin was employed as a loading control. The ability of PCSK9 to induce degradation of the LDLR constructs was quantified using NIH ImageJ software and calculated relative to treatment with pIRES conditioned media. Data are representative of at least three independent experiments. <b>D</b>) PCSK9 reduces cell surface LDLR levels independent of the receptor's CT and TMD. To assess the ability of PCSK9 added exogenously to HEK293 cells expressing the LDLR or its chimeric constructs, transfected cells were treated overnight with empty vector control pIRES-V5 or PCSK9-V5 conditioned media. Subsequently, surface LDLR was quantified by FACS analysis. The values obtained after treatment with PCSK9 are represented graphically relative to treatment with control pIRES. Data are representative of at least three independent experiments. Error bars represent SEM. *, <i>p</i><0.05 (Student's t test).</p
Proteomics analysis of the livers of WT <i>versus Pcsk9<sup>−/−</sup></i> mice.
No full text<p>Proteomics analysis of the livers of <i>Pcsk9<sup>−/−</sup></i> mice. List of membrane proteins identified by quantitative mass spectrometry of livers isolated from <i>Pcsk9<sup>−/−</sup></i> mice and compared to their WT littermates. The proteins listed here include only membrane-bound secretory proteins containing signal peptide/membrane anchors, TMDs, and not exclusively localized in the ER or mitochondrial compartments. “Ratio” values indicate the relate abundance of the protein in <i>Pcsk9<sup>−/−</sup></i> livers <i>versus</i> those of WT mice.</p
The catalytic domain of PCSK9 is required for LRP-1 degradation.
No full text<p><b>A</b>) Chimeric PCSK9 constructs. Schematic representation of PCSK9 and its CHRD coupled to the TMD and CT of LAMP1. The constructs contained C-terminal V5-tags. <b>B</b>) HEK293 cells were transfected with PCSK9 and the CHRD-LAMP1 chimeric constructs. Expression of the PCSK9 constructs was assessed by Western blotting using mAb-V5. The effects of these constructs on the levels LDLR and LRP-1 were determined by immunoblotting with anti-human LDLR and LRP-1 antibodies. LDLR and LRP-1 levels were measured relative to β-actin levels. Data are representative of at least three independent experiments.</p
