HERP-R50H stabilizes Herp endogenous and increases cell toxicity.

<p>(<b>A</b>) Immunoblot of 1AT, HRD1, BiP and Hsp90 protein expression in cell lysates following siRNA-mediated silencing of HERP and HRD1 in Z-IB3 cells. Tunicamycin (Tun) treatment at 2 μg/ml for 24 hours is used as positive control. Quantitative analyses of the immature (black bar graph) and mature (white bar graph) forms of Z-1AT after HERP and HRD1 silencing relative to scrambled control (Scr). Data denote the -fold change in the protein expression of the indicated Z-1AT forms relative to Scr (mean ± S.D., <i>n</i> = 3 independent experiments). In all panels, the * indicate <i>p</i> < 0.05 as determined by two-tailed t-test using Scr as reference. (<b>B</b>) Effect of HRD1 and HERP silencing on steady state levels of Z-1AT in Z-IB3 cells. Cell homogenates were separated into insoluble (left) and soluble (right) fractions and these fractions were then subjected to immunoblot analysis for 1AT (top) and GAPDH (bottom). (<b>C</b>) Percentage of live cells was estimated as described in Materials and methods upon pcDNA, HERP-WT or HERP-R50H overexpression at 48 h after transfection. Data are presented as a ratio relative to control pcDNA empty vector (mean ± SD, n = 3 independent experiments). The asterisk indicates p < 0.05 as determined by two-tailed t-test using pcDNA empty vector as the reference. (<b>D</b>) Immunoblot of Flag tag (upper) and HERPUD1 (Herp) protein expression (lower) in cell lysates following cycloheximide (CHX) treatment. Black arrowhead indicates Flag-Herp expression bands and grey arrowhead indicates endogenous (Endo)-Herp expression bands. Z-IB3 cells were transfected with plasmids encoding the empty vector (pcDNA), HERP-WT, HERP-R50H and HERP-K61R mutation. At 48 h after transfection, 50 μg/ml cycloheximide (CHX) were added and the chase was performed in the absence or presence of CHX for the indicated times. Quantitative analysis of the cycloheximide-chase experiments for the Flag tag (left) and Herp endogenous (right) upon pcDNA, HERP-WT, HERP-R50H or HERP-K61R overexpression is shown. Data shown denote the -fold change relative to <i>t</i> = 0 (mean ± S.D., <i>n</i> = 3 independent experiments). In all panels asterisks indicate <i>p</i> < 0.05 as determined by two-way ANOVA followed by Bonferroni post-test.</p

Genotypes of Herp and HFE variants among ZZ individuals who developed or not liver symptoms.

<p>Genotypes of Herp and HFE variants among ZZ individuals who developed or not liver symptoms.</p

Identification of HRD1 as an important actor in Z-1AT-mediated toxicity in a toxicity assay set up in <i>S</i>. <i>cerevisiae</i>.

<p>(<b>A</b>) Empty Vector, Wild-type (WT) 1AT and Z-1AT expressing cells were grown in inducible media and spotted onto nitrocellulose. After 48 hr of growth at 30°C, the cells were rinsed from the nitrocellulose with distilled water and then immunoblotted for 1AT. (<b>B</b>) Yeast screen methodology summary. The expression of Z-1AT is not toxic in yeast. As a consequence we proposed to identify genes whose presence is necessary for this absence of toxicity, the latter being revealed upon target gene deletion. The Euroscarf library of individual gene deletion mutants is transformed by expression plasmids for either Z-1AT, WT-1AT or the empty vector. Individual colonies are picked and plated in 96-well plates. The growth test is then initiated by inducing the expression of the recombinant 1AT proteins on specific media. Clones with defective growth are selected and the DNA barcode sequence for gene identification purposes. In brief, through this screen, we identified 171 KO strains in which Z-1AT toxicity was slightly changed compared to the empty vector. Then, we have tested these 171 KO strains for the toxicity induced by the WT-1AT expression and found 31 KO mutants in whom Z-1AT was more toxic than the WT-1AT. Finally, only 5 genes displayed a clear human ortholog. (<b>C</b>) Yeast cells transformed with empty vector, WT-1AT or Z-1AT were grown in liquid synthetic complete medium to log phase. Cells were spotted on plates containing SD medium (2% dextrose) to repress 1AT expression or SG medium (2% galactose) to induce 1AT expression, and were incubated at 30°C for 3 days. Shown is fourfold serial dilution starting with equal numbers of cells. Spotting assays for <i>HRD1</i> deletion strain (right panels) and the parental control strain (BY4247 –left panels) are shown.</p

Combined effect of HFE-H63D variant and ERAD impairment in WT and Z-1AT expressing cells.

<p><b>(A)</b> WT and Z-IB3 cells were treated with siRNA-mediated silencing of HERP or HRD1 for 72h and transfected with plasmid encoding HFE-H63D variant or pcDNA. At 48 h after transfection percentage of live cells for each conditions was determined. Data shown denote the -fold change relative to WT-IB3 cells (mean ± S.D., n = 3 independent experiments). In all panels asterisks indicate p < 0.05 as determined by two-tailed t test using WT-IB3 cells or siHERP Z-IB3 cells or siHRD1 Z-IB3 cells as reference. (<b>B)</b> Model for ERAD impairment and HFE-H63D mutation mediated liver toxicity associated with Z-1ATD. The model is adapted from Ordonez <i>et al</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179369#pone.0179369.ref042" target="_blank">42</a>]. An insult resulting from the ERAD default increased the misfolded client proteins into the ER and this insult is effectively buffered by chaperone diffusion in WT-1AT cells. This prevents an UPR “hyperactivation” and the death of cells (‘‘ER stress resolved”). In contrast, in cells that accumulate Z-aggregates, the global ER environment is affected, which impairs chaperon access to misfolded proteins, thereby increasing the cell’s propensity to activate ER stress. An insult resulting from the ERAD default increased the misfolded client proteins, enhanced by the HFE-H63D mutation into the ER, and this insult is not effectively buffered by chaperone in Z-1AT cells. The cells are not anymore able to resolve the ER homeostasis (‘‘unresolved”) and therefore “hyperactivate” the UPR-mediated cell death.</p

HRD1 silencing affects the viability, the traffic and secretion of human cells lines expressing the Z variant.

<p>(<b>A</b>) Viability is measured using GF-AFC Substrate. This compound can enter live cells where it is cleaved by the live-cell protease to release AFC. WT and Z-IB3 cells were treated as described in Material and methods. Quantitative analysis of candidate genes silencing on viability in WT or Z-IB3 cells (mean ± S.D., n = 3 independent experiments). The * indicates p < 0.05 as determined by two-tailed t-test using WT-IB3 cells as reference. (<b>B</b>) Immunoblot analysis of 1AT and Hsp90 protein expression in cell lysates and culture media following siRNA-mediated silencing of the candidate genes HDAC2 and HRD1 in WT and Z-IB3 cells. Traffic of the 1AT glycoprotein through the secretory pathway can be monitored by a change in its migration on SDS-PAGE in response to the processing of ER-acquired N-linked oligosaccharides (the immature form: I) during trafficking through the Golgi to generate the slower migrating, mature glycoform (Mat). The latter is secreted in the serum (secreted form: S) by the cell. Quantitative analyses of the immature, mature, and secreted forms of WT-1AT (black bar graph) and Z-1AT (white bar graph) after candidate genes silencing relative to scrambled control (Scr). Data denote the -fold change in the protein expression of the indicated WT or Z-1AT forms relative to Scr (mean ± S.D., <i>n</i> = 3 independent experiments). In all panels, the * indicates <i>p</i> < 0.05 as determined by two-tailed t-test using Scr as reference.</p

New evidence of a mitochondrial genetic background paradox: Impact of the J haplogroup on the A3243G mutation-0

Impacts on the OXPHOS level, their implications on an individual level, and their impact on the distribution of populations studied within the phylogeny.<p><b>Copyright information:</b></p><p>Taken from "New evidence of a mitochondrial genetic background paradox: Impact of the J haplogroup on the A3243G mutation"</p><p>http://www.biomedcentral.com/1471-2350/9/41</p><p>BMC Medical Genetics 2008;9():41-41.</p><p>Published online 7 May 2008</p><p>PMCID:PMC2409300.</p><p></p