FLASH Knockdown Sensitizes Cells To Fas-Mediated Apoptosis via Down-Regulation of the Anti-Apoptotic Proteins, MCL-1 and Cflip Short

FLASH (FLICE-associated huge protein or CASP8AP2) is a large multifunctional protein that is involved in many cellular processes associated with cell death and survival. It has been reported to promote apoptosis, but we show here that depletion of FLASH in HT1080 cells by siRNA interference can also accelerate the process. As shown previously, depletion of FLASH halts growth by down-regulating histone biosynthesis and arrests the cell cycle in S-phase. FLASH knockdown followed by stimulating the cells with Fas ligand or anti-Fas antibodies was found to be associated with a more rapid cleavage of PARP, accelerated activation of caspase-8 and the executioner caspase-3 and rapid progression to cellular disintegration. As is the case for most anti-apoptotic proteins, FLASH was degraded soon after the onset of apoptosis. Depletion of FLASH also resulted in the reduced intracellular levels of the anti-apoptotic proteins, MCL-1 and the short isoform of cFLIP. FLASH knockdown in HT1080 mutant cells defective in p53 did not significantly accelerate Fas mediated apoptosis indicating that the effect was dependent on functional p53. Collectively, these results suggest that under some circumstances, FLASH suppresses apoptosis

Activation of latent dihydroorotase from \u3ci\u3eAquifex aeolicus\u3c/i\u3e by pressure

Elevated hydrostatic pressure was used to probe conformational changes of Aquifex aeolicus dihydroorotase (DHO), which catalyzes the third step in de novo pyrimidine biosynthesis. The isolated protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC). X-ray crystallographic studies of the isolated DHO and of the complex showed that association induces several major conformational changes in the DHO structure. In the isolated DHO, a flexible loop occludes the active site blocking the access of substrates. The loop is mostly disordered but is tethered to the active site region by several electrostatic and hydrogen bonds. This loop becomes ordered and is displaced from the active site upon formation of DHO-ATC complex. The application of pressure to the complex causes its timedependent dissociation and the loss of both DHO and ATC activities. Pressure induced irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated. However, moderate hydrostatic pressure applied to the isolated DHO subunit mimics the complex formation and reversibly activates the isolated subunit in the absence of ATC, suggesting that the loop has been displaced from the active site. This effect of pressure is explained by the negative volume change associated with the disruption of ionic interactions and exposure of ionized amino acids to the solvent (electrostriction). The interpretation that the loop is relocated by pressure was validated by site-directed mutagenesis and by inhibition by small peptides that mimic the loop residues

FLASH was found in the nucleus co-localized with NPAT.

<p>(<b>A</b>) HT1080 cells (5×10⁶) were fractionated into cytoplasmic (Cyt) and nuclear (Nuc) fractions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032971#s2" target="_blank"><i>Materials and Methods</i></a>). The fractions were analyzed by immunoblotting using antibodies directed against FLASH, NPAT, PARP, HDAC1 and β-tubulin. (<b>B</b>) immunofluorescence co-localization (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032971#s2" target="_blank"><i>Materials and Methods</i></a>) of FLASH and PML or NPAT. HT1080 cells were fixed with cold methanol for 10 minutes, blocked, and incubated with rabbit anti-FLASH and mouse anti-PML antibodies or mouse anti-NPAT antibodies at 4°C overnight. Cells were then washed 3 times and incubated at room temperature for 1 hour with a 1/2000 dilution of the secondary antibodies, Alexa Fluor 594–conjugated anti-rabbit IgG (red) and an Alexa Fluor 488–conjugated anti-mouse IgG antibody (green). The cells were also stained with Hoechst 33342 (blue).</p

Effect of FLASH knockdown on the level of anti-apoptotic proteins.

<p>(<b>A</b>) HT1080 cells were transfected with 3 different FLASH siRNAs for 72 hours. Coilin and the scrambled siRNA served as controls. The intracellular level of FLASH, coilin, MCL-1, histone H3 and the long and short isoforms of cFLIP, cFLIP (L) and cFLIP (S), respectively, were determined by immunoblotting using the corresponding antibodies. β-tubulin served as a loading control. (<b>B</b>) Following the same protocol, MCF-10A cells were transfected with siRNA directed against FLASH or with control siRNA. Cell extracts were prepared 72 hours following transfection and the cell lyates were subjected to immunoblotting using antibodies directed against the indicated proteins.</p

siRNA silencing of FLASH expression.

<p>(<b>A</b>) HT1080 cells were transfected with FLASH siRNA and scrambled siRNA (Control) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032971#s2" target="_blank"><i>Materials and Methods</i></a>). After 72 hours, the extracts of the transfected cells were analyzed by immunoblotting using FLASH antibodies and as a loading control, β-tubulin antibodies. (<b>B</b>) HT1080 cells were transfected with either a scrambled siRNA (left, Control) or a specific siRNA directed against FLASH (right). The cells were fixed with cold methanol for 10 minutes after 72 hours transfection, blocked, and incubated with rabbit anti-FLASH and mouse anti-PML at 4°C overnight. After washing three times, the cells were incubated with the secondary antibodies as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032971#pone-0032971-g003" target="_blank">Figure 3</a>. The cell nucleus was stained with Hoechst 33342. (<b>C</b>) Flow cytometry analysis showed that after FLASH knockdown, cells were blocked in S phase. HT1080 cells were transfected with siRNA against FLASH or scrambled RNAi for 72 hours. The cells were trypsinized, washed with cold PBS, fixed with 70% ethanol, treated with RNase A and stained with 50 µg/ml propidium iodide. The DNA content was analyzed using a Becton-Dickinson FACScan cytofluorometer.</p

The effect of FLASH knockdown on apoptosis was dependent on p53.

<p>(<b>A</b>) HT1080 cells were transfected with scrambled siRNA (siControl), FLASH siRNA (siFLASH) and p53 siRNA (siP53) or co-transfected with both FLASH siRNA and p53 siRNA for 48 hours. Apoptosis was then induced by incubation with 100 ng/ml FasL for an additional 4 hours. Immunoblotting using p53 antibodies showed that p53 was effectively knocked down with siP53 in the presence and absence of siFLASH. Upon stimulation with the FasL, the increase in apoptosis in cells lacking FLASH was abolished in cells depleted of both FLASH and p53. (<b>B</b>) The effect of DNA damage incurred by exposure to adriamycin on the relative intracellular level of p53 and p21. Two isogenic cell lines, HT1080 (wildtype p53) and HT1080-6TG (p53 mutant), were treated with 200 ng/ml adriamycin for the indicated times. The intracellular level of p53 and p21 was determined by immunoblotting. The level of p-Histone H2A.X (Ser139) was used to monitor the progressive DNA damage induced by adriamycin treatment. β-actin served as a loading control. (<b>C</b>) The wild type HT1080 and HT1080-6TG cells (p53 mutant) were transfected, as in panel B, with siRNA against FLASH and the scrambled siRNA (Con) for 72 hours. The intracellular levels of FLASH and MCL-1 were determined by immunoblotting. β-tubulin served as a loading control.</p

Effect of FLASH knockdown on apoptotic progression.

<p>(<b>A</b>) HT1080 cells transfected with control siRNA or with siRNA directed against FLASH were stimulated with mouse anti-Fas antibody (1 µg/ml) following the standard protocol (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032971#s2" target="_blank"><i>Materials and Methods</i></a>) for the indicated times. The cell lysates were subjected to western blotting using anti-FLASH, anti-caspase 8, anti-cleaved caspase 3 and as a loading control, anti-β-tubulin antibodies. (<b>B</b>) HT1080 cells were transfected with two different FLASH siRNAs (FLASH-1 and FLASH-2) and the scrambled siRNA (Control) for 48 hours and then treated with 100 ng/ml FasL for the indicated times. The cell lysates were subjected to immunoblotting using FLASH, PARP, caspase 8, coilin and Fas antibodies. The developed blot was scanned to determine the relative levels of active caspase-8 shown in the bar graph. (<b>C</b>) A time course showing the progression of apoptosis by immunoblotting of PARP and PARP cleavage products in control and FLASH knockdown cells following the procedure outlined in panel B. (<b>D</b>) Immunofluorescence assay of caspase-3 activation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032971#s2" target="_blank"><i>Materials and Methods</i></a>) in HT1080 cells transfected with FLASH or control siRNA for 48 hours with additional 6 hours treatment with 100 ng/ml FasL. (<b>E</b>) Light micrographs of HT1080 cells transfected with FLASH and control siRNA for 72 hours and then stimulated with FasL for 16 hours.</p

The Intracellular level of FLASH decreases during apoptosis.

<p>(<b>A</b>) The proteasome inhibitor MG132 potentiates caspase 8 activation induced by FasL in HT1080 cells. HT1080 cells were treated with the indicated concentration of FasL with or without 10 µM MG132 for 4 hours. The activation of caspase-8 was monitored by immunoblotting of the total cell lysates using caspase 8 antibodies. β-tubulin served as a loading control and p21, a protein with a short half-life, was a control showing that MG132 effectively blocks proteasomal activity. (<b>B</b>) FLASH was down-regulated following induction of apoptosis. HT1080 cells were either pretreated with the vehicle (DMSO) or caspase 3, 8 and 10 inhibitors for 30 minutes and then induced into apoptosis by exposure to 100 ng/ml FasL and 10 µM MG132 for 4 hours. The relative intracellular levels of FLASH, PARP, intact and cleaved, and caspase-3 were determined by immunoblotting. β-tubulin served as a loading control. (<b>C</b>) FLASH was also downregulated following induction of apoptosis in HeLa cells by exposure to 1 µM staurosporine for the indicated times. The cell lysates were analyzed by immunoblotting of FLASH, caspase-9, IKK, coilin and β-actin. (<b>D</b>) Apoptosis was induced by exposure to UV light (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032971#s2" target="_blank"><i>Materials and Methods</i></a>). The cells were harvested 12 hours and 24 hours following a 5 minute UV exposure. The relative levels of FLASH, phospho-p95/NBS1, an indicator of DNA damage, P21 and β-tubulin were determined by immunoblotting. (<b>E</b>) Protein synthesis was blocked by incubating HT1080 cells with 50 µg/ml cycloheximide (CHX) for the indicated times and the relative level of FLASH, coilin, β-actin and p53 was determined by immunoblotting. (<b>F</b>) The relative levels of the same proteins as in panel (E) were determined by immunoblotting following inhibition of RNA transcription by exposure of HT1080 cells to 1 µg/ml actinomycin D for the indicated times.</p

Integrated allosteric regulation in the <it>S. cerevisiae </it>carbamylphosphate synthetase – aspartate transcarbamylase multifunctional protein

<p>Abstract</p> <p>Background</p> <p>The <it>S. cerevisiae </it>carbamylphosphate synthetase – aspartate transcarbamylase multifunctional protein catalyses the first two reactions of the pyrimidine pathway. In this organism, these two reactions are feedback inhibited by the end product UTP. In the present work, the mechanisms of these integrated inhibitions were studied.</p> <p>Results</p> <p>The results obtained show that the inhibition is competitive in the case of carbamylphosphate synthetase and non-competitive in the case of aspartate transcarbamylase. They also identify the substrate whose binding is altered by this nucleotide and the step of the carbamylphosphate synthetase reaction which is inhibited. Furthermore, the structure of the domains catalyzing these two reactions were modelled in order to localize the mutations which, specifically, alter the aspartate transcarbamylase sensitivity to the feedback inhibitor UTP. Taken together, the results make it possible to propose a model for the integrated regulation of the two activities of the complex. UTP binds to a regulatory site located in the vicinity of the carbamylphosphate synthetase catalytic subsite which catalyzes the third step of this enzyme reaction. Through a local conformational change, this binding decreases, competitively, the affinity of this site for the substrate ATP. At the same time, through a long distance signal transmission process it allosterically decreases the affinity of the aspartate transcarbamylase catalytic site for the substrate aspartate.</p> <p>Conclusion</p> <p>This investigation provides informations about the mechanisms of allosteric inhibition of the two activities of the CPSase-ATCase complex. Although many allosteric monofunctional enzymes were studied, this is the first report on integrated allosteric regulation in a multifunctional protein. The positions of the point mutations which specifically abolish the sensitivity of aspartate transcarbamylase to UTP define an interface between the carbamylphosphate synthetase and aspartate transcarbamylase domains, through which the allosteric signal for the regulation of aspartate transcarbamylase must be propagated.</p