18 research outputs found
SEC24A deficiency lowers plasma cholesterol through reduced PCSK9 secretion.
The secretory pathway of eukaryotic cells packages cargo proteins into COPII-coated vesicles for transport from the endoplasmic reticulum (ER) to the Golgi. We now report that complete genetic deficiency for the COPII component SEC24A is compatible with normal survival and development in the mouse, despite the fundamental role of SEC24 in COPII vesicle formation and cargo recruitment. However, these animals exhibit markedly reduced plasma cholesterol, with mutations in Apoe and Ldlr epistatic to Sec24a, suggesting a receptor-mediated lipoprotein clearance mechanism. Consistent with these data, hepatic LDLR levels are up-regulated in SEC24A-deficient cells as a consequence of specific dependence of PCSK9, a negative regulator of LDLR, on SEC24A for efficient exit from the ER. Our findings also identify partial overlap in cargo selectivity between SEC24A and SEC24B, suggesting a previously unappreciated heterogeneity in the recruitment of secretory proteins to the COPII vesicles that extends to soluble as well as trans-membrane cargoes. DOI:http://dx.doi.org/10.7554/eLife.00444.001
Improved survival of aged sensory neurons with nerve growth factor treatment is correlated with reversal of aging-associated decreased Trk a neurotrophin receptor expression, and an increased ratio of Trk A:P75 receptors
Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3
<div><p>Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the best understood cause of dominantly inherited stroke and results from NOTCH3 mutations that lead to NOTCH3 protein accumulation and selective arterial smooth muscle degeneration. Previous studies show that NOTCH3 protein forms multimers. Here, we investigate protein interactions between NOTCH3 and other vascular Notch isoforms and characterize the effects of elevated NOTCH3 on smooth muscle gene regulation. We demonstrate that NOTCH3 forms heterodimers with NOTCH1, NOTCH3, and NOTCH4. R90C and C49Y mutant NOTCH3 form complexes which are more resistant to detergents than wild type NOTCH3 complexes. Using quantitative NOTCH3-luciferase clearance assays, we found significant inhibition of mutant NOTCH3 clearance. In coculture assays of NOTCH function, overexpressed wild type and mutant NOTCH3 significantly repressed NOTCH-regulated smooth muscle transcripts and potently impaired the activity of three independent smooth muscle promoters. Wildtype and R90C recombinant NOTCH3 proteins applied to cell cultures also blocked canonical Notch fuction. We conclude that CADASIL mutants of NOTCH3 complex with NOTCH1, 3, and 4, slow NOTCH3 clearance, and that overexpressed wild type and mutant NOTCH3 protein interfere with key NOTCH-mediated functions in smooth muscle cells.</p> </div
Inhibition of Notch mediated transcription by overexpression of full-length NOTCH3.
<p>Notch expressing cell lines H460 (A) or A7R5 (B–E) were cotranfected with luciferase reporters and either vector or full length NOTCH3 expression plasmids (n = 3 replicate wells per group). In panels A and B, we show effects of NOTCH3, mutants R90C and C49Y, and control vector on ligand stimulation of HES-luciferase. The effects of cotransfection of NOTCH3 on smooth muscle actin-luciferase (C), smooth muscle MHC-luciferase (D), SM22-luciferase (E, left group) and mutant SM22-luciferase (E, right group) are shown in the lower panels. After one day, cells were overlayed with L, Jagged1, or Delta1 expressing fibroblasts to assess the effect of Notch ligand stimulation. Luciferase assays were performed after 24 hours to quantify the level of Notch signaling. All ligand (Jagged1 or Delta1) stimulated activity was significantly suppressed by WT and mutant NOTCH3 transfection for every promoter tested (relative to control; p<0.05; the differences are not marked to simplify the presentation of data). Consistent and significant differences in Notch signaling were observed after coexpression of wildtype or mutant NOTCH3 in for H460 cells transfected with HES-luciferase (A) and for A7R5 cells transfected with all luciferase constructs except the mutant SM22 promoter. There were significant differences between the potency of inhibition of the mutant NOTCH3 proteins in selected groups. Each experiment was conducted three or more times; in each experiment, triplicate wells were analyzed. * indicates differences between NOTCH3 transfected cells versus control cells and # denotes differences between mutant NOTCH3 and wildtype NOTCH3 transfectants (p<0.05). Error bars represent standard deviations.</p
Clearance of mutant NOTCH3 proteins.
<p>293 cells (n = 3 replicate wells per group) were transiently transfected with NOTCH3-luciferase constructs and split into identical 24 well chambers after 24 hours. After another 24 hours incubation, cells were treated with cycloheximide (100 ug/ml) in serum free media to arrest all protein synthesis, and groups of cells were analyzed at time points shown, when cell lysates were prepared and assayed for luciferase enzyme content. (A) The levels of NOTCH3-luciferase activity at the beginning of the time course did not differ significantly between any of the wildtype or mutant combinations. (B) Wildtype NOTCH3 ectodomain was more rapidly cleared than mutant NOTCH3 ectodomains. When cotransfections were performed with wildtype and mutant NOTCH3, proteins were cleared at an intermediate speed. (C) Quantification of a single time point (3 hours) is shown in a bar graph format to facilitate interpretation. The wild type protein is cleared most rapidly. Both CADASIL mutations slow clearance rate significantly. When mutants and wildtype protein were expressed together, there was an intermediate level of stability for R90C/wildtype protein. The C49Y/wildtype mixture was degraded at a similar rate as C49Y protein. Data is representative of three independent experiments. (*Significant compared to wildtype protein, #significant compared to all other groups; p<0.05.) Error bars represent standard deviations.</p
Detergent solubility and formation of inclusions of NOTCH3 expressed in cell culture.
<p>NOTCH3 complexes containing wildtype protein or mutant proteins were tested for detergent solubility. Total protein from cell lines expressing WT or mutant NOTCH3 were sequentially extracted as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044964#s2" target="_blank">methods</a>, using progressively higher strengths of detergents. Cells were grown at low density (A) or in confluent monolayers (B). While WT protein was modestly soluble, the mutant proteins were extractable only in higher detergent concentrations. NOTCH3 protein was detected using antisera from Santa Cruz. The control protein tubulin was readily extractable without detergents. Experiments were performed three times with consistent results. Cell lines were immunostained with the same antisera or with antibodies from R&D, revealing diffuse cell staining with focal accumulations in the perinuclear areas of cells expressing both wildtype and mutant proteins (C). A significant difference in the percentage of cells with perinuclear expression of the protein was notable at low cell density (D) (over 250 cells were counted for each cell line and condition. Results were reproduced three times, and significance was considered p<0.05.</p
Interactions between wild-type and R90C and C49Y mutants of NOTCH3.
<p>(A–C) NOTCH3-NOTCH3 self-association is mediated by the extracellular domain. 293 cells were transiently co-transfected with cDNAs expressing the NOTCH3 extracellular domain tagged with HA (A, B, and C were transfected with WT, R90C, and C49Y, respectively) and cotransfected with myc tagged NOTCH3 ectodomain constructs as shown. The proteins were extracted after 40∼48 hours and immunoprecipitated with HA monoclonal antibody. Co-immunoprecipitates of cells cotransfected with plasmid combinations shown were analyzed by immunoblotting (IB). Quantification of protein bands from four independent experiments failed to show a significant difference in IP complexes with any of the NOTCH3 combinations. (D) Effect of divalent cations on stability of NOTCH3 homophilic interactions and the stability of NOTCH3 homophilic complexes. Coimmunoprecipitation was performed using the HA antibody on lysates of cells transfected with plasmid combinations shown on the right column. Immunoprecipitates were washed extensively with buffers containing calcium or magnesium, and then analyzed by western blotting. All of the HA and myc tagged proteins run on SDS gels at an apparent molecular weight of 175 kDa. We verified the finding of numerous groups that common epitope tags do not mediate protein interaction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044964#pone.0044964-Zhang2" target="_blank">[64]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044964#pone.0044964-Pulipparacharuvil1" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044964#pone.0044964-Nazarenko1" target="_blank">[66]</a> (not shown). All experiments were performed more than 6 times.</p
NOTCH3 ectodomain interacts with both NOTCH1 and NOTCH4 ectodomains.
<p>Interactions between NOTCH3 (WT and mutants) with NOTCH1 or NOTCH4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044964#pone.0044964-Small1" target="_blank">[41]</a> were tested after cotransfection of plasmid combinations shown. Protein from transfected cells were evaluated by immunoblotting (input) and by immunoprecipitation with V5 monoclonal antibody followed by immunoblotting (IP:V5). Each of the NOTCH3 ectodomain constructs coprecipitated with both V5-tagged NOTCH1 or NOTCH4 proteins, which migrate with apparent molecular weights of >280 kDa and 180 kDa, respectively. All experiments were performed three times.</p