A138V, p53M237I, p53R248W, and p53R273P but not the p53R175H mutant protein. Importantly, we show that Def directly interacts with CAPN3 in the nucleoli and determines the nucleolar localisation of CAPN3, which is a prerequisite for the degradation of p53 in the nucleolus. Furthermore, we find that Def is modified by phosphorylation at five serine residues: S50, S58, S62, S87, and S92. We further show that simultaneous phosphorylations at S87 and S92 facilitate the nucleolar localisation of Capn3 that is not only essential for the degradation of p53 but is also important for regulating cell cycle progression. Hence, we propose that the Def-CAPN3 pathway serves as a nucleolar checkpoint for cell proliferation by selective inactivation of cell cycle-related substrates during organogenesis.Digestive organ expansion factor (Def) is a nucleolar protein that plays dual functions: it serves as a component of the ribosomal small subunit processome for the biogenesis of ribosomes and also mediates p53 degradation through the cysteine proteinase calpain-3 (CAPN3). However, nothing is known about the exact relationship between Def and CAPN3 or the regulation of the Def function. In this report, we show that CAPN3 degrades p53 and its mutant proteins p53 During organogenesis, cell proliferation is controlled not only by many known regulatory networks but also by the newly identified Def-CAPN3 nucleolar protein degradation pathway. The nucleolus is primarily known as the subcellular organelle for the biogenesis of the ribosomal small and large subunits in eukaryotic cells. However, it is also increasingly evident that many nucleolar proteins also play essential roles in other biological processes. Here, we show that Digestive organ expansion factor (Def), a nucleolar protein, determines the nucleolar localisation of the cysteine proteinase CAPN3, and they form a complex that degrades the tumor suppressor protein p53, an essential regulator of the cell cycle, in the nucleolus. Furthermore, we show that Def is modified by phosphorylation at five serine residues and that Def phosphorylation modification facilitates the nucleolar localization of Capn3, which is also important for regulating cell cycle progression. Hence, we propose that the Def-CAPN3 pathway serves as a nucleolar checkpoint of cell proliferation by selective inactivation of cell cycle-related substrates during organogenesis, revealing a novel biological role for the nucleolus. Arabidopsis . Si. Sidef-/S87,92E) . QuantitS87,92E) .def-/-. We previously showed that the knockdown of p53 expression only partially rescued pancreas development in def-/-, which led us to propose that Def promotes digestive organ development in part through the p53 pathway. To further confirm this hypothesis, we co-injected p53-MOATG  and p53-MOspl (to block the maturation of zygotic p53 mRNA) [def-/- and its siblings (WT or heterozygotes)  than in def-/- (2.3%). Similarly, the pecentages of PCNA-positive hepatocytes were higher in the morphants (15%) than in def-/- (10%)  and obtained the def-/-p53M214K double mutant. Surprisingly, analysis of the liver development using an fabp10a probe showed no significant difference in liver sizes between the def-/- single and def-/-p53M214K double mutant , as previously reported [Because p53 is a target of the Def-Capn3 pathway, one scenario is that loss of function of p53 might rescue liver development in 53 mRNA)  into defzygotes) . The injzygotes) . Bhmt an/- (10%) . These re mutant , indicatreported .The nucleolus is a peculiar subnuclear structure that houses several hundred to a thousand proteins. Increasingly, data have shown that the nucleolus is not only a site for the assembly of ribosomal subunits but is also highly responsive to extrinsic or intrinsic stress signals to regulate cell behaviour. This latter function is largely due to the great diversity of functional proteins localised within the nucleolus. In this report, we showed that Def and CAPN3 form a complex in the nucleolus and that Def determines the nucleolar localisation of CAPN3. We further showed that the function of the Def-CAPN3 protein degradation pathway is regulated by Def phosphorylation at five Ser residues: S50, S58, S62, S87, and S92. Specifically, simultaneous phosphorylation at S58 and S62 or at S87 and S92 is not only important for Def in the mediation of p53 degradation in the nucleolus but is also essential for normal transition from G1 to S and from G2 to M during cell proliferation. Our findings firmly specify a key role of the nucleolar Def-CAPN3 protein degradation pathway in the cell cycle progression during organogenesis, thus establishing a novel biological role for the nucleolus—the “old factory.”R175H mutant protein is CAPN3 insensitive. This is consistent with our previous report on the insensitivity of p53R143H to Def-mediated p53 degradation in zebrafish. It is probable that the R175 mutation is close to the CAPN3 recognition motif (188–198 AA) in p53, and that the R175H mutation alters the conformation in the CAPN3 recognition motif in p53 [R175H resistant to the CAPN3 enzymatic activity. This implies that patients who carry the p53R175H mutation can potentially accumulate a high level of non-degraded p53 in the nucleoli. Indeed, by analysing the distribution of p53 proteins in three different types of breast cancer cells, we found that p53R175H appeared to accumulate in the nucleolar fraction  and identified the mutation at p53R175 as one of the hotspots for cancer mutation   . Further samples , and p53 samples .m proteins, specifically in the liver, we showed that simultaneous phosphorylation of the S58 and S62 pair and the S87 and S92 pair regulates Def function, particularly its role in the mediation of nucleolar p53 degradation, cell cycle progression, and nucleolar localisation of Capn3b. Thus, we propose that the function of Def phosphorylation is probably to provide a microenvironment that is favourable for the enzymatic activity of CAPN3. Alternatively, phosphorylation is important for Def to interact with other nucleolar partners that are essential for the activity of CAPN3. In summary, we propose that Def not only serves as a carrier to ferry CAPN3 to the nucleolus but also acts as a scaffold protein to provide a setting for the enzymatic activity of CAPN3 on the target protein. Interestingly, protein sequence alignment showed that S58 and S62 are conserved among zebrafish, mice, and humans, which implies that these two sites are also likely to be important in mammals. In contrast, S87 and S92 in zebrafish are not conserved in mammals . The likely scenario is that during evolution, S87 and S92 in lower vertebrates was converted to E91 and E96 in mammals, suggesting that in the lower vertebrates, these two amino acids need to be modified by phosphorylation to fulfil their biological function, whereas in mammals, these two amino acids mimic the constitutive active form of the zebrafish Def as shown in the Tg transgenic fish. Regarding the role of Def in liver development, we argue that because Def not only teams up with Capn3 to form the Def-Capn3 pathway but also is a component of the ribosomal small subunit processome (for the biogenesis of ribosome), both of these functions of Def might contribute to promoting digestive organ growth, including liver. This would explain the partial rescue of the digestive organ development in def-/- when the expression of p53 is knocked down, because there might be other substrates of the Def-Capn3 pathway, which are essential to regulate liver development, or the function of Def in ribosome biogenesis is also necessary for the growth of digestive organs, including liver.Protein function is often modulated by the modification of phosphorylation status . ThroughMany questions remain to be answered about this novel nucleolar protein degradation pathway. For example, it appears that one or more sites exist within the 190–377 AA region in Def that are also modified by phosphorylation. One impending task is to determine such site(s) in the region. It is now clear that Def is phosphorylated at multiple sites, so we must ask whether they are modified by single or multiple kinases and what these kinase identities are. Next, does Def phosphorylation form a regulatory feedback loop? If so, how is it regulated in response to different stimuli? Does Def-CAPN3 work with other partners to mediate p53 degradation in the nucleolus? In addition to p53, are there any other targets of the Def-CAPN3 pathway? Insights into these questions will be instrumental in understanding the biological function of the Def-Capn3 pathway.All animal procedures were performed in full accordance to the requirement by “Regulation for the Use of Experimental Animals in Zhejiang Province.” This work was specifically approved by the Animal Ethics Committee in the School of Medicine, Zhejiang University .def-/- (defhi429) [Tg(fabp10a:def)-1 was generated as described [Tg(fabp10a:defm) lines, each fabp10a:defm was cloned into a pDB739 vector (modified from pminiTol2 obtained from Dr. Stephen Ekker) to obtain the fabp10a:defm-miniTol2 plasmid. Each fabp10a:defm-miniTol2 plasmid DNA was injected together with Tol2 transposase mRNA [def_Fw1644 (5ʹ-CCAGGTGTGCAGTAAAACCATT-3ʹ) and def_Rv2048 (5ʹ-GGCAACCCGTAGAAGATCAGA-3ʹ). Zebrafish were raised and maintained in accordance with the standard procedures [Zebrafish line efhi429)  was as defhi429) . Tg. After centrifugation, the supernatant was collected and incubated with Ni-NTA agarose beads (QIAGEN), washed twice, and eluted using 500 mM imidazole. The eluent was desalted by ultrafiltration with a pre-equilibration buffer .GST-CAPN3 was cloned into the expression vector pGEX-6P-1. His-Def1-379 was obtained by PCR amplification using forward (5ʹ CGCGGATCCATGCATCATCACCACCACCATGGCAAACGCGGGAGCCGGAGC-3ʹ) and reverse (5ʹ-GCTCTAGAGTCACCCTCGAGGAGGCTGA-3ʹ) primers and cloned into pET30a. E. coli BL21 (DE3) was used for protein expression. The cells were sonicated in lysis buffer . The GST-CAPN3 lysate was incubated with GST-beads (Novagen) overnight and washed five times with lysis buffer. His-Def1-379 was retained by Ni-NTA agarose beads (QIAGEN), eluted by 500mM of imidazole, and desalted with lysis buffer. The GST beads were incubated with the purified His-Def1-379 for 8 h. The beads were washed six times and eluted with SDS lysis buffer.mRNA was synthesised in vitro using mMESSAGE mMACHINE Kit (Ambion) according to the manufacturer’s instructions. A total of 0.2 ng of mRNA was injected into one-cell stage zebrafish embryos to overexpress the protein of interest. The embryos were first deyolked and lysed in SDS lysis buffer supplied with 1×Complete Protease Inhibitor Cocktail . The protein samples were immediately used for western blot analysis or stored at -20°C. For western blot analysis, rabbit polyclonal antibody against Def (using the CLRLPDSPQRPEPDS peptide as antigen) and Capn3b was generated by Hangzhou Hua An Biotechnology Company (China). Anti-Myc tag antibody  was purchased from Clontech; GFP , p53 ,  from Santa Cruz; CAPN3 (No. 38963), NPC , and p53  from Abcam; CAPN3 goat polyclonal antibody (No. COP-080048) from Cosmo Bio; GAPDH  from Epitomics, and GAPDH , β-actin (R1207-1) from Hua An.2, 1 mM dithiothreitol, 50 mM Tris-HCl, pH 7.9) supplied with 1×Complete Protease Inhibitor Cocktail  and 0.2% NP40. Ten units of CIP were added to 40 μl supernatant and incubated at 37°C for 1 h. The reaction was stopped by adding 13 μl of SDS sample buffer before SDS-PAGE.For CIP (NEB) treatment, the deyolked embryos were lysed in Tris lysis buffer -EGFP-D14-(6×His)-pCS2+. Then, 24 μg of plasmid DNA per plate was used to transfect 293T cells cultured in a 10 cm plate (three dishes were used). At 36 h after transfection, the total protein was extracted and (6×His)-EGFP-D14-(6×His) was purified using Ni-NTA agarose beads (Qiagen) according to the manufacturer’s instructions.6×His tagged EGFP-D14 was derived from EGFP-D14-pCS2+ using the primers His-EGFP  and His-D14 . The amplified fragment was cloned into pCS2+ pre-digested with 3 pieces and de-colored in 25 mM of ammonium bicarbonate/50% acetonitrile buffer. The samples were then incubated with 10 mM of DTT in 50 mM of ammonium bicarbonate at 56°C for 1h followed by 55 mM of iodoacetamide in 50 mM of ammonium bicarbonate in the dark for 45 min, followed by overnight digestion with trypsin  in 25 mM ammonium bicarbonate at 37°C. Peptides were extracted from gel with buffers containing 5% trifluoroacetic acid and 50% acetonitrile by two rounds of ultrasonication. The liquid was freeze-dried by SpeedVac, and the peptides were resolubilised in 0.1% formic acid and filtered with a 0.45-μm centrifugal filter.The SDS-PAGE gel containing approximately 50 μg (6×His)-EGFP-D14-(6×His) of protein was cut into 2- to 3-mm6 ions. The 20 most intense ions from each MS scan were isolated, fragmented, and measured in the linear ion trap, with MSA neutral losses of m/z 98, 49, and 32.6. The CID normalised collision energy was set to 35.For the MS analysis, the resuspended peptides were analysed by LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) coupled online to an Easy-nLC 1,000 (Thermo Fisher Scientific) in the data-dependent mode . Peptides were separated by reverse phase LC with a 75 μm (ID) × 150 mm (length) analytical column packed with C18 particles of 5 μm in diameter. The mobile phases for the LC contain buffer A  and buffer B , and a linear gradient of buffer B from 3% to 30% for 90 min was used for separation. All MS measurements were performed in the positive ion mode. Precursor ions were measured in the Orbitrap analyser at 240,000 resolution (at 400 m/z) and a target value of 10TM software version 1.3. The proteome sequences for Danio rerio  from unitprot were used for a database search with the mass tolerance set to 0.05 Da. The maximum number of missed cleavages was set at two, with a minimum peptide length of six and a maximum peptide length of 144 amino acids. The false discovery rate was set at 0.01 for peptide and protein identification. Serine, threonine and tyrosine phosphorylation, cysteine carbamidomethylation, and methionine oxidation were included in the search as variable modifications.The data were analysed using a prerelease version of Thermo Scientific Proteome Discovererfabp10a, trypsin, and def probes [For WISH, digoxigenin  was used to label the f probes . WISH waf probes .The embryos were fixed in 4% PFA for 1 h at room temperature. After washing in PBST, the embryos were mounted with 1.5% agarose dissolved in 30% sucrose PBST and then equilibrated overnight in 30% sucrose PBST at 4°C. The blocks were mounted with OCT compound (Sakura). The sections were cut serially to a 12-μm thickness and collected on poly-L-lysine coated glass slides . The slides were used immediately for immunofluorescence or stored at -80°C.The cryosections were rehydrated with two washes of 1xPBS and then permeated with PBS plus 0.2% Triton X-100 for 5 min. After a brief wash with PBS, the sections were blocked by 20% goat serum in PBS plus 0.5% BSA. After a brief wash with PBS plus 0.5% BSA, the sections were incubated overnight with primary antibody at the desired concentration diluted in PBS plus 0.5% BSA at 4°C. After three washes with PBS plus 0.5% BSA, sections were incubated with secondary antibodies (1:400) and DAPI (1:500) in PBS plus 0.5% BSA for 1 h. After three washes with PBS plus 0.5% BSA, the sections were finally mounted in 80% glycerol and covered with coverslip for image acquisition. Def antibody (1:200) , antibodTUNEL assay was carried out following the procedures previously described  with theEdU  was injected into the anterior region of the yolk between the heart and liver of the embryos at 2.5 or 3 dpf. After incubation at 28.5°C for 2 h, the embryos were fixed in 4% PFA for 2 h before proceeding to cryo-section. Incorporated EdU was detected by Alexa Fluor 488 Azide .2, and 0.5 mM DTT). After centrifugation at 218 g for 5 min at 4°C, the nuclear pellet was resuspended in S1 solution  and cleared by centrifugation over a cushion of S2 solution . The nuclei were resuspended in S2 solution and sonicated  for six 10-s bursts  at 40% amplitude. The sonicated sample was layered onto S3  and centrifuged at 3,000 g for 10 min. The supernatant contained nucleoplasmic fractions. The nucleoli were resuspended with 0.5 ml of S2 solution, followed by centrifugation at 1,430 g for 5 min at 4°C.HepG2 cell nucleoli were isolated as previously described . Brieflyg for 5 min at 4°C, the nuclear pellet was washed twice in buffer A. The pellet was then resuspended in zfS1  and sonicated for five 8-s bursts  at 50% amplitude. The sonicated sample was layered onto zfS2  and centrifuged at 1,800 g for 10 min. The supernatant contained nucleoplasmic fractions.The zebrafish liver nucleoli were isolated as previously described  with minHuman cells were digested with trypsin and washed twice with PBS, then stained with PI  according to the manufacturer’s instructions. Zebrafish embryos were raised to 8 dpf and fixed with 0.5% PFA at 4°C for 0.5 h. After washing with PBS, liver bud labeled by DsRed signal was dissected under a fluorescence microscope  using ultra micro tweezers; the non-liver tissues sticked to the liver were carefully removed in bright field using a fine needle. Liver cells were dissociated by treatment with 0.1% trypsin at 37°C for 20 min and were washed with PBS. Cells were stained with PI and subjected to flow cytometry analysis of the cell cycle using a BD FACS Calibur flow cytometer.t test. * p < 0.05; ** p < 0.01; *** p < 0.001; N.S., no significant difference. To cope with the relative small sample sizes, we used the quartile boxplot to present our data on liver size measurement shown in Statistical analyses were performed with the Student’s S1 Data(XLSX)Click here for additional data file.S1 Fig(A) Both human p53 (hu-p53) and zebrafish p53 (zf-p53) contain two conserved putative CAPN3 recognition motifs, highlighted with colored shade. (B) Western blot of His-CAPN3 eluted by 20, 50, 100, 200, and 500 mM imidazole from the Ni-NTA agarose beads. His-CAPN3 was immunoblotted by a rabbit polyclonal antibody against CAPN3.(TIF)Click here for additional data file.S2 Fig(A) Western blot of CAPN3C129S and CAPN3C129S-ΔNOLS in total protein extract  and the nucleolar fraction (NO). 293T cells were transfected with respective plasmids and harvested at 72 h. (B) Western blot of hu-Def showing the knockdown of hu-Def by the hu-def–specific siRNAs. GAPDH: loading control. (C) Co-IP analysis of the interaction between HA-tagged Capn3bC120S and Myc-tagged zebrafish Def, Def_S50, Def_S58,62A, or Def_S87,92A mutant proteins in 293T cells. Total protein was extracted from co-transfected cells at 72 h after transfection and was then incubated with Myc-beads. Western blot was performed with anti-HA and anti-Myc antibodies.(TIF)Click here for additional data file.S3 FigE. coli. Total protein crude extract (CE) was treated with (CE+CIP @ 37°C) or without (CE @ 37°C) CIP at 37°C for 1 h. To purify (6xHis)-EGFP-D14-(6xHis), total protein crude extract was mixed with the Ni-NTA agarose beads followed by washing (wash 1) and eluting with 250 mM of imidazole (elution 1 and elution 2). Upper panel: Coomassie blue staining (CBB), lower panel: western blot of (6xHis)-EGFP-D14-(6xHis) using an antibody against zebrafish Def. Protein samples are as shown. FT, flow through.(6xHis)-EGFP-D14-(6xHis) was expressed in (TIF)Click here for additional data file.S4 Fig Mass spectra showing the precursor ions of the identified peptides bearing phosphorylated amino acid residues S50, S58, and S62 (A), and S87 and S92 (B) in (6xHis)-EGFP-D14-(6xHis).(TIF)Click here for additional data file.S5 FigThe phosphorylated residues  were highlighted.(TIF)Click here for additional data file.S6 Fig(A) Table listed the transgenic lines obtained. Tg(fabp10a:def) was obtained as described [def_Fw1644 and def_Rv2048 was used in PCR to genotype transgenic fishes.  WISH analysis of def expression pattern in different transgenic fishes as shown using the def probe. The liver is highlighted with a red arrow.escribed . Two ind(TIF)Click here for additional data file.S7 Figdef-/- mutant and different transgenic lines in the def-/- background at 4 dpf as shown. DAPI was used to stain nuclei (in blue). Scale bar: 5 μm.Co-immunostaining of Def or its mutant protein (in red) and nucleolar marker Fibrillarin  in wild-type (WT), (TIF)Click here for additional data file.S8 Fig(A) Representative images showing TUNEL assay for detecting the apoptotic cells in the liver and other organs of 4-dpf embryos in different genotypes as indicated. White arrows indicate the apoptotic cells observed in the epidermis and neural tube. Asterisk indicates the site of the embryonic liver. WT embryo section pretreated with DNase I (WT+DNase I) was used as the positive control. Scale bar: 50 μm. (B) Summary of TUNEL assay in different genotypes.(TIF)Click here for additional data file.S9 Fig(A–E) 3-dpf WT embryo sections were co-stained with an anti-PCNA antibody, anti-Fabp10a antibody (to show hepatocytes), and DAPI (to stain nuclei). The high magnification of the region outlined by white box in (A) is presented to show PCNA signal (B), Fabp10a signal (C), DAPI staining (D), and merged image (E). Hepatocytes harboring distinct brilliant foci (highlighted with a white arrow in B) were considered to be cells at S-phase, whereas cells with even distributed PCNA signal to be at non-S-phase. Scale bar: 5 μm.(TIF)Click here for additional data file.S10 Fig(A–D) A representative image showing the liver bud in a Tg reporter fish (A) and a dissected liver bud from the same reporter fish . Each transgenic fish line was crossed to the Tg background and raised to 8 dpf. Livers of different genotyped fish were dissected under a fluorescent microscope, and cells were dissociated. Dissociated cells were co-stained with DAPI (to stain nuclei) (B) and the DsRed fluorescence (C) for counting the number of DsRed-positive cells (hepatocytes) (D). In each case, more than 94% of the cells were found to be DsRed-positive cells. Scale bar: 100 μm. (E) Flow cytometry analysis of the liver cells isolated from 8-dpf zebrafish of different genotypes as indicated. Representative graph from three independent experiments is shown here. Statistical analysis of the flow cytometry data is shown in (E) are provided in (TIF)Click here for additional data file.S11 Fig+/+ or HCT116-p53-/- cells at 24 h after treatment with ctrl-siRNA, capn3-siRNA, or hu-def-siRNA. Representative graph from three independent experiments is shown. Statistical analysis of the flow cytometry data is shown in Graph showing the flow cytometry analysis of the HCT116-p53(TIF)Click here for additional data file.S12 Fig(A) Western blot of p53 showing the knockdown of p53 by p53-specific morpholino mixes (ATG-MO plus spl-MO) in zebrafish embryos at 3 dpf. β-actin: loading control. (B) Representative images of P-H3 and PCNA immunostaining in 3-dpf old def-/- embryos injected with p53-specific morpholino mixes (ATG-MO plus spl-MO). Scale bar: 100 μm. Hepatocytes are marked by the Bhmt antibody (images for P-H3 staining) or Fabp10a antibody (images for PCNA staining). Nuclei are stained by DAPI. Liver area is outlined by a dashed line.(TIF)Click here for additional data file.S13 Fig(A) Western blot analysis of p53, Def, Fib, and NPC in the cytoplasm (CP), nucleoplasm (NP), and nucleolar (NO) fractions extracted from three different breast cancer cell lines: MCF-7 (p53WT), SK-BR-3 (p53R175H), and JIMTI (p53R248W). The basal levels of p53 in these cell lines were very different. (B) Histogram showing the p53 NO/NP ratios in three different cancer cells by grey-value analysis of the band intensity shown in (A). (C) p53R175 is one of the hotspots for cancer mutation, with 1,260 tumour samples harbouring the R175 substitution mutation. Scale bar on the top represents the amino acid position in p53. (D) p53R175H mutation ranks first among the p53 substitution mutations in cancer samples. (E) p53R175H is not specific for any cancer type when compared with other p53 mutations A138V, M237I, and R248W. All four of these p53 mutations were observed more frequently in large intestine, breast, haematopoietic and lymphoid, and ovary tumour samples. The y-axis indicates the percentage of tumour of a specific tissue in total tumour samples of the p53 A138V, R175H, M237I, or R248W substitution recorded. Data shown in (C–E) were retrieved from the Catalogue of Somatic Mutations in Cancer database (http://cancer.sanger.ac.uk/cosmic). Underlying data for (B), (C), and (E) are provided in (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.


VPS34 is reported to activate S6K1 and is implicated in regulating cell growth, the mechanisms of which remain elusive. Here, we describe novel mechanisms by which VPS34 upregulates mTOR/S6K1 activity via downregulating TSC2 protein and activating RheB activity. Specifically, upregulation of VPS34 lipid kinase increases local production of ptdins(3)p in the plasma membrane, which recruits PIKFYVE, a FYVE domain containing protein, to ptdins(3)p enriched regions of the plasma membrane, where VPS34 forms a protein complex with PIKFYVE and TSC1. This in turn disengages TSC2 from the TSC1/TSC2 heterodimer, leading to TSC2 ubiquitination and degradation. Downregulation of TSC2 promotes the activation of RheB and mTOR/S6K1. When VPS34 lipid kinase activity is increased by introduction of an H868R mutation, ptdins(3)p production at the plasma membrane is dramatically increased, which recruits more PIKFYVE and TSC1 molecules to the plasma membrane. This results in the enhanced TSC2 ubiquitination and degradation, and subsequent activation of RheB and mTORC1/S6K1, leading to oncogenic transformation. The role played by VPS34 in regulating mTOR/S6K1 activity and cellular transformation is underscored by the fact that the VPS34 kinase dead mutant blocks VPS34-induced recruitment of PIKFYVE and TSC1 to the plasma membrane. This study provides mechanistic insight into the cellular function of VPS34 in regulating oncogenic transformation and important indications for identifying VPS34 specific mutations in human cancers. The mammalian target of rapamycin complex 1 (mTORC1) has been established as a critical molecule in the regulation of protein translation, cell cycle progression and cell proliferation . It is bTSC1 and TSC2 are the tumor-suppressor genes mutated in tumor syndrome TSC (tuberous sclerosis complex). Over the past decade, their gene products, TSC1/TSC2 heterodimer, have emerged as a critical integrator of growth factor, nutrient and stress signals to control protein synthesis, cell growth and other cellular processes [rocesses . It is nrocesses –9. Overerocesses . While trocesses –13. The rocesses . Howeverrocesses . Moreoverocesses .Vacuolar protein sorting 34 (VPS34), class III PI3K, mediates nutrient signaling to mTORC1, leading to the activation of S6 Kinase 1 (S6K1) and regulation of protein synthesis –19. InhiPIK3CA gene [PIK3CA gene has been found in human cancers [in vitro and in vivo [Class I PI3K plays critical roles in cell growth and its tumorigenic activity is activated by somatic point mutations , 22. p113CA gene . A high  cancers . Cancer- cancers . E542 an cancers . The mut in vivo , 28. Whi in vivo . We repo in vivo . HoweverWe searched for VPS34 mutations with potential gain of lipid kinase activity by performing a sequence alignment of VPS34 and p110α of class I PI3K. We observed that while the H1047 hot spot of p110α was not conserved in VPS34 (V867), the site H1048 of p110α was conserved with VPS34 (H868) Figure . We geneWe next tested whether upregulation of VPS34 affected mTOR activity and its downstream signaling pathways. Figure We next investigated the relationship between VPS34 and TSC1/TSC2 heterodimer. Figure It has been reported that the FYVE domain containing protein, PIKFYVE, specifically recognizes ptdins3)p and targets cytosolic proteins to ptdins(3)p-enriched membranes and mediates mTOR activation , 33. We p and tarGiven that TSC1 is required for the stability of TSC2 , we hypoRheB is inactivated by GTP hydrolysis catalyzed by TSC2. We next tested whether VPS34-mediated downegulation of TSC2 resulted in activation of RheB. Figure Cyclin E plays a critical role in cell cycle progression from G1 to S phase. Western blot data showed that cyclin E expression was higher in H868R_07 cells than WT_118 and Vector cells Figure . Cyclin To test whether overexpression of either VPS34-WT or VPS34-H868R induces cellular transformation, two VPS34-WT clones (WT_108 and WT_118) and two VPS34-H868R clones (H868R_07 and H868R_08) with different levels of VPS34 expression Figure  were ranWe next asked whether overexpression of VPS34-WT or VPS34-H868R induced tumor formation in mice. Subcutaneous injection of H868R_07 cells into severely compromised immune-deficient (SCID) mice resulted in the formation of solid tumors after 1-2 weeks Figure  and 6F. We next used pharmacological approaches to confirm that cell growth and transformation induced by VPS34 occurred via mTORC1/S6K1 pathway. Figure Class I PI3Ks, which are composed of p110α, the catalytic subunit, and p85, the regulatory subunit, are important in regulating proliferation and tumorigenesis . MechaniPIKFYVE targets many cytosolic proteins to ptdins(3)p-enriched membranes and mediates mTOR activation , 33 and in vitro and in vivo [VPS34 has been studied extensively in the context of endocytic sorting, and has been implicated in regulating nutrient signaling , 19, 38. in vivo . The onc in vivo , 40, 41.NIH3T3 cells and COS-7 cells were purchased from American Type Culture Collection (ATCC) and used within six months after cells were ordered. These cell lines were not authenticated in our laboratory. Rapamycin, 5-Bromo-2′-deoxyuridine (BrdU) and cycloheximide were obtained from Sigma. PF-4708671 was purchased from Selleckchem. MG132 was purchased from CalBiochem. Antibodies directed against Myc-tag (9E10), Flag-tag, and HA-tag were obtained from Covance. Antibodies directed against VPS34 (cat#4263), VPS15 (cat#14580), Beclin 1 (cat#3495), pT389-S6K1 (cat#9234), S6K1 (cat#2708), pT37/46-4EBP1 (cat#2855), 4EBP1 (cat#9644), pS235/236-S6 (cat#4858), S6 (cat#2217), pErk1/2 (cat#4370), Erk1/2 (cat#4695), pS473-Akt(cat#4060), Akt (cat#4685), RheB (cat#13879), TSC1 (cat#6935), TSC2 (cat#4308), BrdU (cat#5292), and Cyclin E (cat#4129) were purchased from Cell Signaling Technology. Fluorescently tagged antibodies were obtained from Invitrogen. The class III PI3K lipid kinase ELISA kit was purchased from Echelon Biosciences. The RheB activation kit was purchased from Abcam. 4-6 weeks old SCID mice were ordered from National Cancer Institute (NCI), NIH .Wild-type pcDNA3-Myc-hVPS34 was a kind gift from Dr. Jonathan Backer of Albert Einstein College of Medicine. The kinase dead VPS34 construct, pcDNA3-Myc-hVPS34-D747N, N748K (designated VPS34-KD), and pJ4HA-VPS34 constructs were generated as described previously , 31, 42.5 cells per well and transiently transfected with empty vector or plasmids encoding VPS34 or VPS34 mutants. Cells were harvested 48h post transfection and WCL were subjected to immunoprecipitation. The immunoprecipitated VPS34 proteins bound on beads were used for the kinase assay. The absorbance was read at 450nm on an ELISA plate reader. OD values were plotted against Log ptdins(3)p values to generate a standard curve using a sigmoidal dose response. Ptdins(3)p production (pmol) for each enzymatic reaction was determined by interpolation from the standard curve.VPS34 lipid kinase activity was assessed using a class III PI3K ELISA kit (Echelon Biosciences) according to the manufacturer's instruction. This assay is a competitive ELISA in which the signal is inversely proportional to the amount of ptdins(3)p produced. Briefly, COS-7 cells were seeded in 6-well plates at a density of 1×107 cells). Following washing with ice-cold PBS, the cells were lysed in 0.5 ml 1X ice-cold Assay/Lysis Buffer. Whole cell lysates (WCL) obtained from Vector, WT_118, and H868R_07 were subjected to the assay. WCL were incubated with 1 μL anti-active RheB mouse monoclonal antibody and 20 μL protein A/G agarose bead slurry at 4°C for 1 hour with gentle agitation. Beads were pelleted by centrifugation, washed 3 times with Lysis Buffer and then subjected to SDS-PAGE analysis. RheB was detected by Western blot analysis using a rabbit antibody directed against RheB-GTP. The samples of positive and negative controls were generated according to manufacturer's protocol.The RheB activation assay was performed using a RheB activation kit  according to the manufacturer's protocol. Briefly, Vector, WT_118 and H868R_07 cells were cultured in 10 cm dishes until they reached 90% confluence . 48h post-transfection, WCL were subjected to immunoprecipitations using anti-TSC2 or IgG control antibody. Ubiquitinated endogenous TSC2 was detected by Western blot with anti-HA antibody. The immunoprecipitated endogenous TSC2 was detected by Western blot using anti-TSC2 antibody for assessing TSC2 degradation. The protein levels of Myc-tagged VPS34-WT and Myc-tagged VPS34-H868R in the WCL were detected by Western blot using anti-Myc antibody. For the detection of Flag-tagged TSC2 ubiquitination, COS7 cells were transiently co-transfected with plasmids encoding empty vector, VPS34-WT or VPS34-H868R plus Flag-TSC2 and HA-ubiquitin. 48h post-transfection, Flag-tagged TSC2 was immunoprecipitated from WCL using an anti-Flag antibody. Ubiquitinated Flag-TSC2 was detected by anti-HA antibody. Cycloheximide protein chase experiment was performed to monitor degradation of TSC2. Cells stably expressing vector and VPS-34 (H868R_07) were incubated with cycloheximide at 50 μg/ml for 2, 4 and 6h or left untreated. After incubation, WCL was collected and western blotting was performed to detect the levels of TSC2 using anti-TSC2 antibody. TSC2 accumulation assay was performed using MG132, a potent, reversible and membrane-permeable inhibitor of proteasome. Stably expressing H868R_07 cells were treated with MG132 at 5μM for 24h or left untreated. WCL was subjected to western blot analysis to detect TSC2 expression levels.The experiments were performed according to manufacturer's instructions . For flow cytometric cell cycle analysis of BrdU incorporation, one million cells were harvested by centrifugation, and incubated with fresh serum free media containing BrdU (0.03 mg/ml) for 30 min at 37°C. Cells were rinsed, fixed in cold 70% ethanol, incubated with 1.5M HCl for 30 min at room temperature and rinsed with PBS three times. Cells were blocked in incubation buffer (0.5% BSA in PBS) for 10 min, and incubated with mouse anti-BrdU primary antibody at a 1:200 dilution in incubation buffer for 1 hr at room temperature. Cells were washed with PBS and incubated with anti-mouse Alexa Fluor 488 conjugated secondary antibody for 30 min. Cells were washed again and re-suspended in 1 ml PBS, and analyzed on a BD FACS Calibur flow cytometer. Data are presented in the form of histogram plots and quantified as percentage of BrdU-positive cells that are present in S phase of cell cycle.GraphPad Prism and Microsoft Excel software were used for statistical studies. Statistical significance was determined by Student's t-test . Data are expressed as mean ± SEM.7 cells were injected subcutaneously into each dorsal site of SCID mice using a 27-gauge needle on syringe. Tumors were allowed to grow 4 weeks. At the end of the experiments, tumors were excised from mice and fixed in 10% neutral buffered formalin (Sigma). Fixed samples were sent to Histoserve, Inc  for paraffin sectioning and hematoxylin and eosin (H&E) staining for histopathological evaluation of tumors. H&E stained slides were scanned using panoramic MIDI scanner . The images were acquired at a 20X magnification, and relevant areas were converted into a TIF format for the generation of figures. Stained tissue sections were analyzed by a board-certified veterinary pathologist.All animal experiments were performed in accordance with animal protocol #WO-2013-08, which was approved by the United States Food and Drug Administration (FDA) Center for Biologics Evaluation and Research (CBER), Institutional Animal Care and Use Committee, in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals. Four-to-six weeks old male severely compromised immune-deficient (SCID) mice purchased from the National Cancer Institute (NCI) were allowed at least one week to acclimatize to the animal facility prior to the initiation of experiments. Mice were randomly assigned into 3 experimental groups . Stably expressing cell lines were cultured in DMEM media supplemented with 10% calf serum (CS). At 80% confluence, cells were harvested by trypsinization, washed twice with PBS and counted. Cells were then centrifuged at 1000 rpm for 5 min and cell pellets were re-suspended in 400 μL PBS. 1×10


The YAP and TAZ transcriptional coactivators promote oncogenic transformation. Elevated YAP/TAZ activity has been documented in human tumors. YAP and TAZ are negatively regulated by the Hippo tumor suppressor pathway. The activity and stability of several Hippo pathway components, including YAP/TAZ, is regulated by ubiquitin mediated protein turnover and several ubiquitin ligase complexes have been implicated in human cancer. However, little is known about the deubiquitylating enzymes that counteract these ubiquitin ligases in regulation of the Hippo pathway. Here we identify the DUB3 family deubiquitylating enzymes as regulators of Hippo pathway activity. We provide evidence that DUB3 proteins regulate YAP/TAZ activity by controlling the stability of the E3 ligase ITCH, the LATS kinases and the AMOT family proteins. As a novel Hippo pathway regulator, DUB3 has the potential to act a tumor suppressor by limiting YAP activity. Transcriptional co-activators Yes-Associated Protein 1 (YAP1) and transcriptional coactivator with PDZ-binding motif (TAZ) mediate the activity of the Hippo signaling pathway in control of cell proliferation and in tumor progression . IncreasA number of different mechanisms have been reported by which ubiquitin-mediated protein turnover regulates YAP/TAZ activity. The best studied of these involves the targeting of YAP and TAZ for destruction by the βTrCP/SCF ubiquitin ligase system, as a consequence of LATS1/2 mediated phosphorylation of YAP and TAZ ,11. DestThe activity of ubiquitin ligases in promoting protein turnover can be counteracted by deubiquitylating enzymes that remove ubiquitin moieties from proteins. Evidence is emerging that deubiquitylating enzymes may act as oncogenes and tumor suppressors –22. UsinIn this report, we present evidence implicating the family of DUB3 deubiquitylating enzymes in control of Yap/TAZ activity. DUB3 proteins act by regulating the stability of the E3 ubiquitin ligase ITCH, the Hippo pathway core kinases LATS1 and LATS2, as well as the family of AMOT proteins, which play a scaffolding role in DUB3-mediated regulation of YAP.Antibodies to NEDD4 (Cat #2740), ITCH (#12117), SMURF1 (#2174), phosphor-YAP , YAP (#4912), YAP/TAZ (#8418), phospho-LATS1 , LATS1 (#9153), LATS2 (#13646), specific K48 and K63 ubiquitin and Myc Tag (#2278) were from Cell Signaling Technology . HA (#sc-7392) and YAP (sc#101199) antibodies were from Santa Cruz Biotechnology . Anti-AMOT was from ABNOVA . Anti-Flag antibody and anti-Actin, HA-, Myc- and Flag-conjugated beads were from Sigma-Aldrich, St Louis, MO, USA). DUB3 antibody (Cat#PA-5-44961) was obtained from Invitrogen .ATACGGATCCACCATGGACTACAAGGATGACGATGACAAGGACTACAAGGATGACGATGACAAGGAGGACGACTCACTCTACTT and ATCAGAATTCTCACTGGCACACAAGCAGAG. The PCR product was sequenced and encodes the USP17L17 isoform of DUB3. The C89S mutant was made by PCR as previously described . Th. Th33]. ITCH-mediated ubiquitylation has been shown to promote the turnover of several Hippo components, such as LATS kinases and AMOT proteins ,18,34. OThese findings present an apparent conundrum: DUB3 interacts with ITCH and affects ITCH ubiquitylation and stability, and higher ITCH expression should increase turnover of LATS and AMOT. However, despite the increase in ITCH protein, LATS and AMOT were stabilized as a consequence of DUB3 expression. Reciprocally, they were destabilized as a consequence of DUB3 depletion. We therefore considered the possibility that DUB3 might directly regulate the turnover of AMOT and LATS proteins.We examined the effect of DUB3 depletion on AMOT ubiquitylation. In control cells, DUB3 was recovered by co-precipitation with AMOT, providing evidence for physical interaction . DepletiAMOT proteins have been proposed to play a scaffolding role, mediating the interaction between ITCH and YAP, to promote YAP ubiquitylation . AMOT prNext we asked whether depleting the LATS1/2 kinases would block the effects of DUB3 on YAP levels. DUB3 depletion led to an increase in YAP levels . DepletiOur findings provide evidence that the DUB3 deubiquitylating enzyme acts at several levels to regulate the activity of the Hippo tumor suppressor pathway . The table summarizes the number of mismatches between the individual siRNAs and shRNAs with the family members.The NCBI RefSeq Gene collection comprises 30 USP17L (USP17L1 through USP17L30) gene entries: 25 are annotated as protein coding genes , while four are annotated as pseudogenes , and one (USP17L23) is annotated as protein coding gene, but not linked to either a RefSeq mRNA or protein sequence. ((PDF)Click here for additional data file.S2 FigControls for the efficacy of shRNAs and siRNAs on DUB3 mRNA expression. (A) HEK293T cells were transfected with a control vector or with independent shRNAs targeting DUB3. (B) HEK293T cells were transfected with a control siRNA (scrambled sequence) or independent siRNAs targeting DUB3. Expression of DUB3 mRNA was measured by quantitative real-time RT-PCR (qPCR) using two independent pairs of primers. Primer pair #1 was used subsequently for measuring DUB3 expression. GAPDH mRNA was used for normalization. Data represent the average of 3 independent experiments ± SD. * indicates p <0.05  comparing the test and control samples.(PDF)Click here for additional data file.S3 FigHEK293T cells were transfected with a vector expressing DUB3 or the catalytically-inactive C89S mutant form or with an empty vector as a control. mRNA expression of YAP, TAZ, ITCH, AMOT, AMOT L1, AMOT L2, LATS1, LATS2 and DUB3 was measured by qPCR. GAPDH mRNA was used as a normalization standard and TBP was used as an independent control. Data represent the average of 3 independent experiments ± SD.(PDF)Click here for additional data file.S4 FigG12V and depleted of p53 and p16 (BJp53kd/p16kd/HRas) were virally transduced and selected to express DUB3, its inactive C89S mutant form or an empty control vector. Cells were plated for 36h before being harvested for immunoblotting. Blots were probed with antibodies against Flag, DUB3, ITCH, phospho-LATS1/2 (T1079/1041), LATS1, AMOT L1, phospho-YAP S127, YAP and actin. (B) BJp53kd/p16kd/HRas cells were virally transduced and selected to stably express either a control vector or independent shRNAs against DUB3 and processed as in (A). (C) BJp53kd/p16kd/HRas cells were transfected to express the luciferase reporters together with siRNAs to deplete DUB3 or with a control scrambled siRNA. Data represent the average of three independent transfection experiments ± SD.(A) BJ fibroblast cells expressing hTert and H-Ras(PDF)Click here for additional data file.S5 Figp53kd/p16kd/HRas cells were seeded for 24h before being transfected with DUB3-specific siRNAs or scrambled controls. Transfected cells were fixed after 36h and stained with YAP antibody . qPCR was used to confirm the depletion of DUB3. (B) Representative images of BJ cells stained with anti-YAP treated as described in panel A. YAP localization was scored as: less YAP in the nucleus compared to the cytoplasm (e.g. white arrows); equal in cytoplasm and nucleus (e.g. blue arrows); and YAP higher in the nucleus (e.g. red arrows). (C) DUB3 depletion caused a significant shift towards nuclear YAP .(A) BJ(PDF)Click here for additional data file.S6 FigHEK293T cells were transfected with a vector expressing DUB3 or the inactive C89S mutant form or with an empty vector as a control. mRNA expression of NEDD4, ITCH and SMURF1 was measured by qPCR. GAPDH mRNA was used for normalization and TBP was used as an independent control. Data represent the average of 3 independent experiments ± SD.(PDF)Click here for additional data file.S7 FigImmunoprecipitation assays. HEK293T cells were treated with 5μM MG132 to block proteasome function 24h before cells were harvested for IP by lysing in PLC buffer, or a modified RIPA buffer containing 10% glycerol. Lysates were immunoprecipitated with anti-ITCH or anti-DUB3 antibodies or isotype-matched control antibodies. Blots were probed with anti-ITCH, anti-DUB3 antibodies. Endogenous ITCH was recovered in the DUB3 IP, but DUB3 was not recovered in the ITCH IP.(PDF)Click here for additional data file.S8 FigHEK293T cells were transfected to express Myc-tagged ITCH along with ubiquitin and siRNA targeting DUB3 or a scrambled control siRNA. Transfected cells were treated with 5μM of MG132 and 5μM of Lactacystin overnight before being subjected to immunoprecipitation with anti-Myc. Blots were probed with antibodies against DUB3, actin, and the Myc epitope to detect Myc-tagged ITCH and with antibodies specific to ubiquitin linked at lysine residues K48 and K63.(PDF)Click here for additional data file.S9 Fig(A) HEK293T cells were transfected to express HA-tagged AMOT along with ubiquitin and DUB3 siRNA or a scrambled siRNA. Transfected cells were treated with 5μM of MG132 and 5μM of Lactacystin overnight before being subjected to immunoprecipitation with anti-HA. Blots were probed with antibodies against HA, DUB3, actin and specific lysine residues K48 and K63 of ubiquitin. (B) HEK293T cells were transfected to express HA-tagged AMOT together with ubiquitin and Flag-DUB3 or Flag-DUB3 C89S. Blots were probed with antibodies against Myc, HA, DUB3, actin.(PDF)Click here for additional data file.S10 Fig(A) HEK293T cells were transfected to express Flag-DUB3, its inactive mutant C89S or a control vector. Transfected cells were treated with 5μM of MG132 overnight before being subjected to immunoprecipitation with anti-Flag. Blots were probed with antibodies against Flag, LATS2, HA and actin. (B) HEK293T cells were transfected to express HA-LATS2 and myc-ubiquitin along with Flag-DUB3, its inactive mutant C89S or a control vector. Cells were treated with 5μM of MG132 overnight before being subjected to immunoprecipitation with anti-HA. Blots were probed with antibodies against Myc, LATS2, HA, DUB3 and actin.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Cs gene of A/J mice has been linked to low activity of the enzyme in skeletal muscles. The aim of the study was to test this hypothesis and examine effects of low citrate synthase (CS) activity on palmitate metabolism in muscle cells. Results of the study showed that carriers of the wild type (WT) Cs  had higher CS activity (p < 0.01) than carriers of the A/J variant (B6.A-(rs3676616-D10Utsw1)/KjnB6 and A/J mouse strains) in the heart, liver and gastrocnemius muscle. Furthermore, the recombinant CS protein of WT showed higher CS activity than the A/J variant. In C2C12 muscle cells the shRNA mediated 47% knockdown of CS activity reduced the rate of fatty acid oxidation compared to the control cells. In summary, our results are consistent with the hypothesis that H55N substitution causes a reduction in CS activity. Furthermore, low CS activity interferes with metabolic flexibility of muscle cells.The H55N polymorphism in the  Mitochondria play a key role in metabolic health and functioning of skeletal muscle ,2. ImpaiCs gene of A/J mice, i.e. the H55N substitution  which is predicted to affect protein function palmitate (PerkinElmer—NEC534050UC) with pH adjusted to 7.4. In the second series of experiments with mixed substrate 5.5 mM glucose was added to the media. Following 2-h incubation period, 1 ml of the medium was transferred to 15 ml tube, the cap of which housed a Whatman (GF/B) filter paper disc that had been pre-soaked with 200 μl of 1M KOH. [14C]O2 trapped in the media was then released by acidification of media using 60% (vol/vol) perchloric acid and gentle agitation of the tubes at 37°C for 2 h. Afterwards, the filter paper disc was removed from the cap and placed into 2 ml of scintillation liquid. The remaining cells were subsequently washed with 2 ml of phosphate buffered saline and lysed in 1 ml of 1 M NaOH before being transferred into 4 ml of scintillation liquid. Samples were then subjected to liquid scintillation counting . The protein content of samples was measured in triplicates using Bradford assay.For assessment of palmitate metabolism C2C12 cells were incubated in the media containing 20 mM Hepes, 140 mM NaCl, 16.1 mM KCl, 5.1 mM MgSOImmunohistochemistry was applied for assessment of changes in cellular levels of ceramide. C2C12 muscle cells were grown and differentiated on extracellular matrix coated 96 well plates. Prior to initiating the assessment cells were starved in substrate limited medium  for 24 h. The cells were then incubated in substrate enriched media, i.e. DMEM supplemented with 0.8 mM palmitate, 0.5 mM carnitine, 100 nM insulin with 5.5 mM glucose (P+G experiments) or without it (P experiments) for 3 h, 12 h and 18 h. Afterwards, C2C12 muscle cells were fixed for 10 min with 4% formaldehyde and permeabilized with 0.4% Triton X-100 in PBS for 6 min, washed, and blocked by 20% goat serum  in PBS for 30 min. Then monoclonal antibody against ceramide  was diluted at 1:10 in 0.025% Tween 20 in PBS solution and applied onto cells with gentle agitation on a rocker for 24 h at 4°C. Afterwards, the secondary antibody anti-mouse coupled to Alexa fluor 555  diluted at 1:500 in 0.025% Tween 20 solution was applied for 90 min. To stain nuclear DNA, C2C12 muscle cells were incubated in 4,6’-diamidino-2-phenylindole (DAPI)  at 1:1000 in PBS for 30 min covered from light on a rocker. C2C12 muscle cells were observed using Zeiss observer Z1 imaging confocal system  and the images were captured using Axiocam . Fluorescence was quantified between treatments as a single in focus plane was acquired. Using ImageJ (v1.49) a single coloured image was split into grey/black images from their red and blue fluorescence and an outline was drawn around each grey area and the mean fluorescence was measured, along with black background readings. The total corrected cellular fluorescence (TCCF) = integrated density–(area of selected cells × mean fluorescence of background readings), was calculated. Ceramide mean TCCF was then normalised against the mean TCCF of nuclear DNA in the same field of view.2, 0.5 mM dNTP Mix , 5 mM DTT, 150 ng of Random primers and 200 units of SuperScript™ III Reverse Transcriptase. Real time PCR was performed using Roche Lightcycler 480 II  and Multiplex Taqman assays for Cs as a target gene and β-Actin as a reference gene in each sample. Three μL of cDNA was added to 10 μL of LightCycler® 480 Probe Master (Roche), 0.2 μL of TaqMan® probe , 0.2 μL of forward and reverse primers (20 μM) each and 1 μL of mouse β-Actin probe dye VIC-MGB . The mouse Cs intron spanning primers were designed using Universal Probe library software and purchased from Sigma-GenoSys (Forward primer: 5’-GGAAGGCTAAGAACCCTTGG-3’ and Reverse primer: 5’-TCATCTCCGTCATGCCATAGT-3’) and the corresponding UPL probe (UPL probe #100) were used. The results were analysed using LightCycler® 480 software 1.5 and Cs was normalised to β-Actin and presented as a ratio (Ratio = (1 + ECs)Cs]-Ct[ / (1 + Eβactin)β-actin]-Ct[) [Cell samples were homogenised in 1 ml of ice cold TRIZOL Reagent  and RNA extracted using chloroform and isopropanol as described previously . 2 μg of-actin]) .3VO4. Protein concentration was determined in triplicate using the Bradford Assay . For immunoblotting, the samples were loaded on 10% polyacrylamide gel, separated using SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membrane. Then membranes were washed with Tris buffered saline (TBS) containing 0.1% (vol/vol) Tween-20 (TBS-T buffer) before being blocked with 5% (wt/vol) non-fat milk in TBS-T buffer. For analysis of proteins carrying His tags, membranes were incubated for 3 h at 4°C with the anti-polyHistidine antibody . For CS protein analysis, membranes were incubated overnight with the CS antibody . Analysis of cell signalling was performed using the overnight incubation antibodies from Cell Signalling Technology . The primary antibodies against AMPK (#2532), phospho-AMPKα (Thr172) (#2531), ACC (#3662), phospho-ACC (ser79) (#3661), β-actin (#4967), p44/44 MAPK (#9102), phospo-p44/42 MAPK (Thr202/Tyr204) (#9101), p38 MAPK (#9212), phospo-p38 MAPK (Thr180/Tyr182), mTOR (#2972) and phospo-mTOR (Ser2448) (#2971). The exposure to the primary antibodies was followed by 2-h incubation with HRP-conjugated secondary antibody  and detection using ECL detection reagent  and Fluor-SMax Imager for immunobloting . The images were quantified using ImageJ  software.As previously described, cell an–1·cm–1 was used to assess the maximum CS activity (Vmax) at 412 nm during the first 2 min of the reaction. The assay was carried out at room temperature (~21°C), and CS from porcine heart was used as a standard  for assay calibration. For β-hydroxyacyl-coenzyme (CoA) dehydrogenase (HAD) assay, the molar extinction coefficient used was 63,000 M-1cm-1 for NADH at 340 nm. The reaction reagent consisted of 100 mM tetrasodiumpyrophosphate, 0.23 mM NADH, 0.24 mM acetoacetyl CoA with pH adjusted to 7.3. The 1000 μL of reaction reagent included 20 μL of muscle homogenate. Citrate concentration was determined using the standard colorimetric-enzyme assay kit  and the plate reader . For sample preparation, cells were lysed, homogenized and centrifuged at 13,000 g for 20 min using 10 kDa molecular weight cut off filters  to generate filtrates for citrate measurements. For cytochrome C oxidase (COX) measurements, the pellets from the filters were removed by centrifugation at 10,000 g for 10 min and re-constituted in 300 μl enzyme dilution buffer of standard COX assay kit . The protein concentration of was always assessed using Bradford assay.CS activity was measured as previously described . The reaAll data analysis was performed using Prism 3.0 software. Analysis of variance (ANOVA) followed by t-tests with Bonferroni correction for multiple comparisons were used to assess differences between the means.p < 0.0001) in all the tissues. For the heart and liver, BALB and B6 mice showed consistently higher CS activity than B6/B6.A, B6.A and A/J mice. In the gastrocnemius muscle B6.A and A/J strains had lower CS activity than BALB and B6 strains, while CS activity for A/J strain was also lower (p < 0.05) compared to B6/B6.A mice.Data on CS activity in the heart, liver and gastrocnemius muscle of the mice are presented in p < 0.0001) and was higher (p < 0.05) for B6 construct compared to the A/J construct . CS protein content was also halved and there was ~47% reduction (p < 0.001) in CS activity for Cs shRNA cells compared to Con shRNA cells (p < 0.05) increase in cell index though peak values of this index did not differ between Cs shRNA and Con shRNA cells (p < 0.05) in Con shRNA cells compared to Cs shRNA cells  basal oxygen consumption (p < 0.05) proton production (p < 0.05) maximal oxygen consumption, spare respiratory capacity (p < 0.05) non-mitochondrial respiration  in Cs shRNA cells compared to Con shRNA in the presence of 5.5 mM glucose  or with 5.5 mM glucose (P+G experiments). Cellular fluorescence for ceramide was normalised to DAPI fluorescence which reflected nuclear DNA content in this analysis (p < 0.01–0.001) afterwards and was greater (p < 0.05) in P+G compared to P experiments. For P experiments, ceramide levels were greater (p < 0.05) in Cs shRNA cells compared to Con shRNA cells after 18 h. The same tendency of the difference (p = 0.068) between Cs shRNA cells and Con shRNA cells was observed in P+G experiments.analysis . There wanalysis . HoweverCs allele. Furthermore, the introduction of this H55N substitution into the Cs cDNA caused a reduction in enzyme activity of the recombinant CS protein. In agreement with our second hypothesis, shRNA mediated Cs knockdown led to low rates of oxidative phosphorylation, increased reliance on anaerobic glycolysis and a high proliferation rates of C2C12 muscle cells. We have also observed higher rates of ceramide accumulation in muscle cells with low CS activity compared to the control cells during incubation with palmitate. It appears that a 47% reduction in CS activity is associated with alterations in cellular metabolism indicating reduced metabolic flexibility which becomes significant when cells are exposed to substrate rich media containing palmitate.The aims of the study were twofold. Firstly, we tested the hypothesis that the A/J variant of the H55N polymorphism is associated with low CS activity. Secondly, we examined if low CS activity interferes with functioning and metabolism of muscle cells. Our results support both these hypotheses. Indeed, mouse strains carrying the A/J variant of H55N polymorphism showed lower CS activity in all tissues measured compared to mouse strains expressing the wild type Cs gene, i.e. B6.A and B6/B6.A mice. Interestingly, however, the variation in CS activity between the strains was greater for the gastrocnemius muscles than for the heart and liver. Skeletal muscle is a malleable tissue which shows differences in fibre type composition and mitochondrial content between homologous skeletal muscles of various mouse strains [Firstly, CS activity was measured in different tissues of mice in order to examine the hypothesis concerning the link between H55N polymorphism and CS activity. The results are in agreement with previous findings showing lower CS activity in the gastrocnemius muscles of A/J mice compared to other mouse strains ,19. The  strains ,20. ThesCs, Shmt2, Lrp1, Zbtb39, BC089597, Gls2, Apon, Stat2 and Pan2 carry non-synonymous SNPs between the two strains. Some of those genes (Shmt2 and Gls2) may even play a role in mitochondrial function. Therefore, to eliminate possible confounding effects of other genes we compared the enzymatic activity between the A/J and wildtype variants of Cs following the overexpression of the recombinant CS protein in transformed RosettaBlue (DE3) cells. This substitution caused 15–50% reduction in CS activity of the recombinant CS protein. There might be some degradation of CS protein when the overexpression was carried out at 30°C [It is important to note that the congenic region of B6.A strain contains a number of genes that are polymorphic between B6 and A/J strains. In addition to  at 30°C ,21. Neveet al.[An inborn defect in the CS enzyme is hypothesized to cause low rates of fatty acid oxidation in the cell cultures generated from muscle biopsies of patients with type 2 diabetes ,11. Howeet al.. It appeet al.,24. ReduWe have also examined effects of low CS activity on mitochondrial markers and signalling proteins including AMPK which is involved in mitochondrial biogenesis. The results of these measurements do not show any differences between C2C12 cells with reduced and normal levels of CS. Thus, reduction in aerobic capacity of C2C12 cells with low CS activity is not due to low mitochondrial content and cells do not experience energy stress when incubated in standard media for cell culturing. It is likely that anaerobic glycolysis can compensate for alterations in aerobic metabolism under the basal conditions when cells are not subjected to high metabolic demands.A limitation of this study is that the utilised homogenization process abolishes mitochondrial integrity. Thus it is not possible to distinguish between cytosolic and mitochondrial CS activity or citrate accumulation. This should be taken into account when assessing these results. Nevertheless, observations of whole-cell physiological changes and metabolism remain valid.Muscle cells are often exposed to exercise or the physiological consequences of high fat diet which induces significant stress in these cells. Defective skeletal muscle cell metabolism is seen as a major contributor to the pathology of obesity and insulin resistance, particularly the inability to upregulate lipid oxidation in response to a high glucose and palmitate overload .Prolonged incubation with palmitate, which is the most abundant fatty acid, leads to apoptosis and impairment of mitochondrial function in various types of muscle cells ,27. PromIn summary, the H55N polymorphism affects CS activity in tissues of mice carrying A/J variant of Cs gene. Low activity of CS results in an impairment in aerobic ATP synthesis and low rates of fatty acid oxidation in muscle cells. These alterations in lipid metabolism render the muscle cell less resistant to metabolic stress as induced by high extracellular levels of palmitate.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.S10 Table(PDF)Click here for additional data file.S11 Table(PDF)Click here for additional data file.S12 Table(PDF)Click here for additional data file.S13 Table(PDF)Click here for additional data file.S14 Table(PDF)Click here for additional data file.S15 Table(PDF)Click here for additional data file.S16 Table(PDF)Click here for additional data file.S17 Table(PDF)Click here for additional data file.S18 Table(PDF)Click here for additional data file.S19 Table(PDF)Click here for additional data file.S20 Table(PDF)Click here for additional data file.


O-methyltransferase (COMT) have not been studied together to determine their effects on smokers. The aim of this study was to investigate the effects of the interaction between the CYP 2A6 and COMT genes on smoking behavior in young Taiwanese men.Although some effects of gene–gene interactions on nicotine–dopamine metabolism for smoking behavior have been reported, polymorphisms of cytochrome P450 (CYP) 2A6 and catechol-CYP 2A6 and COMT genes as well as urinary nicotine and urinary cotinine levels were determined. The odds ratio for starting smoking was significantly lower in subjects carrying a CYP2A6 low activity/variant COMT rs4680 genotype than in those possessing a CYP2A6 wild-type/variant COMT rs4680 genotype . Comparisons of Fagerstrom Test for Nicotine Dependence (FTND), Physiological Cigarette Dependence Scale (PCDS), and Cigarette Withdrawal symptoms (CWS-21) among the smokers with different CYP2A6/COMT polymorphisms were not significantly different. The adjusted urinary nicotine concentrations were not significantly different between the two groups carrying different genotypes. The adjusted urinary cotinine level was significantly different between the COMT rs4680 wild-type group and COMT rs4680 variant group  and between the COMT rs4680 wild-type/COMT rs165599 variant group and COMT rs4680 variant/COMT rs165599 variant group .A self-report questionnaire regarding smoking status was administered to 500 young men. Polymorphisms of the COMT gene and the interaction between the CYP 2A6 and COMT genes affect smoking status in young Taiwanese men.These findings suggest that a single nucleotide polymorphism (rs4680) of the The online version of this article (doi:10.1186/s12993-017-0127-2) contains supplementary material, which is available to authorized users. Tobacco smoking is a multi-factorial behavior with both genetic and environmental determinants . GeneticNicotine is the major psychoactive ingredient in tobacco, and it modulates dopamine activity in the midbrain, which contributes to the development and maintenance of rewarding behaviors such as smoking . SmokersCYP) 2A6 gene, located on chromosome 19q12-q13.2, consists of nine exons. It is involved in producing a 494-amino-acid protein that oxidizes coumarin, nicotine, and tobacco-specific nitrosamines [CYP2A6 is the primary human enzyme involved in nicotine metabolism [CYP2A6 catalyzes the C-oxidation of nicotine to the inactive metabolite cotinine and the subsequent conversion of cotinine into trans-3′-hydroxycotinine [CYP2A6 is the most studied enzyme involved in both adult and adolescent smokers [CYP2A6 is different from that among other ethnic groups [CYP2A6 gene that affect smoking status could be different from those in other ethnic groups.The cytochrome P450 (c groups . TherefoO-methyltransferase (COMT) gene is located on chromosome 22q11.21, has eight exons, and produces a 271-amino-acid protein that metabolizes catecholamines [COMT gene, which encodes a key enzyme involved in the metabolic inactivation of dopamine, has been associated with nicotine dependence [COMT, rs165599, has been related to the response to bupropion therapy for smoking cessation [The catechol-olamines . Low enzpendence . Anotheressation . Howeveressation , 11. ThuDRD2) TaqIB and monoamine oxidase A (MAOA) affected smoking intensity in young men [COMT and MAOA [MAOA and CYP2A6 [CYP2A6 and the nicotine acetylcholine receptor gene [CYP 2A6 and DRD2 TaqIA [CYP2A6 and COMT together have not been explored, and we hypothesized that a single polymorphism as well as an interaction of the two genes could be involved in smoking status.Smoking addiction is currently a significant social problem in Taiwan . Nonetheoung men . Those fand MAOA , 16, MAOd CYP2A6 , CYP2A6 tor gene , 19, andD2 TaqIA . HoweverCYP2A6*1A (wild-type), CYP 2A6*1B (polymorphism of faster nicotine clearance) [CYP2A6*4C (the most studied polymorphism of decreased nicotine metabolism) [COMT Val/Met (rs4680) [COMT rs165599 [CYP 2A6 and COMT genes and their association with smoking risk in young Taiwanese men.In women not using oral contraceptives, nicotine and cotinine clearance is 13 and 24% higher, respectively, than in men . Sex difearance) , CYP2A6*(rs4680) , and COMrs165599  were detThis cross-sectional study was advertised to all students at both Chang Jung Christian University and Central Taiwan University of Science and Technology. The volunteers contacted the authors of the study, and convenience samples were then screened for eligibility at the health centers of the two universities. All study subjects provided written, informed consent at the beginning of study, and the study was approved by the review board of Chang Jung Christian University (CJCU-99004) and Central Taiwan University of Science and Technology (CTUST-99016). The study was conducted in accordance with Good Clinical Practice procedures and the Declaration of Helsinki.We administered a self-report questionnaire to all of the study subjects –27. The CYP2A6 gene have been identified [CYP2A6*1A) and two highly prevalent (>10%) variants of CYP2A6*1B (approximately 45%) [CYP2A6*4C (approximately 15%) [In this study, to determine the genotypes of the variants of interest, total genomic DNA was isolated from blood cells using a blood DNA isolation kit . Five milliliters of whole blood with EDTA as an anticoagulant were required. Approximately 40 alleles of the entified . Howeverely 45%)  and CYP2ely 15%) , which hCYP2A6 were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) [BstU-I as the restriction enzyme, one (1332-bp) and two (291 and 1041-bp) fragments were obtained for CYP2A6*1A and CYP2A6*non-1A, respectively. Using the restriction enzyme Bsu36-I, three fragments  were observed for CYP2A6*1A or CYP2A6*1B. Four fragments  were found for CYP2A6*4C. Positive controls were run for each of the genotyping assays. The DNAs of CYP2A6*1A/*1A, CYP2A6*1A/*1B, CYP2A6*1B/*1B, CYP2A6*1A/*4C, and CYP2A6*1B/*4C, which have been found in Taiwanese and Chinese populations, were identified by DNA sequencing [The polymorphisms of CR–RFLP) . We desiquencing . Additioyme, one 332-bp anCOMT rs4680, the forward primer 5′-CTGTGGCTACTCAGCTGTG-3′ and reverse primer 5′-CCTTTTTCCAGGTCTGACAA-3′ were used to amplify a 169-bp fragment [N1a III as the restriction enzyme, three , four , and five  fragments were obtained for the G/G, A/A, and G/A genotypes, respectively [Msp I as the restriction enzyme, one (301-bp), two (166 and 135-bp), and three  fragments were obtained from the A/A, G/G, and A/G genotypes, respectively [For fragment . Using Nectively . For COMTo evaluate the effects of genetic polymorphisms on the metabolism of nicotine and cotinine, urinary nicotine and cotinine levels of smokers were measured. Gas chromatography–mass spectrometry (GC/MS) was performed as previously described , with thCYP2A6 genotypes consisted of wild-type (*1A/*1A), high -activity (*1A/*1B and *1B/*1B), and low-activity  genotypes. The COMT rs4680 genotypes consisted of wild-type (G/G) and variant (G/A and A/A). The COMT rs165599 genotypes consisted of wild-type (A/A) and variant (A/G and G/G) genotypes. To assess the interaction between the effects of CYP2A6 and COMT gene polymorphisms on smoking behaviors, multiple models were used to analyze the CYP2A6, COMT rs4680, and COMT rs165599 data: (1) single gene (model 1: CYP2A6) or single SNP ; (2) 2 SNPs (model 3: COMT rs4680 and COMT rs165599); and (3) multiple genes (models 4–7). We evaluated the effect of the interactions between different gene combinations on smoking status , as appropriate, was applied to compare quantitative data. A P value of <0.05 was defined as statistically significant for each analysis  as 1 to subjects carrying the wild-type. The Mantel–Haenszel Chi square test was utilized to calculate the ORs and their 95% confidence intervals (CIs) .We recruited a total of 500 men aged 20–25 years. Analysis of the questionnaires revealed that there were 219 never smokers, 261 current smokers, and 20 ever smokers. Due to the small sample size , the 20 ever smokers were excluded from further study. Mean age did not differ between the 261 current smokers and the 219 never smokers . The other 14 ORs were not statistically significant (P = 0.078–0.988).As shown in Table The average number of cigarettes per day among the 261 current smokers was 10. There were 127 heavy smokers and 131 light smokers (data for three smokers were missing). With light smokers as the reference group, 15 ORs for heavy smoking were not statistically significant   and between the COMT rs4680 wild-type/COMT rs165599 variant group and the COMT rs4680 variant/COMT rs165599 variant group . The other comparisons were not statistically significant.Of the 261 current smokers, 122 subjects provided urine samples for the determination of nicotine and cotinine levels by GC/MS. The values for adjusted nicotine concentration and adjusted cotinine concentration were not normally distributed in either genotype group. The SD was too high: near the mean value or even greater than the mean value. Therefore, the Mann–Whitney U test or Kruskal–Wallis test with Bonferroni adjustment was used for comparison of the values of adjusted nicotine concentration and adjusted cotinine concentration between groups carrying different genotypes. As shown in Table All subjects in this study were young adult university students. Alcohol and drug abusers were excluded. Therefore, the effects of age, education status and alcohol and drug interactions on smoking were absent.COMT rs4680 does not agree with Hardy–Weinberg equilibrium, consistent with reports that Asiatic individuals have Hardy–Weinberg disequilibrium of the COMT rs4680 polymorphism [COMT rs4680, the subjects carrying the low-activity genotype of CYP2A6 have a 0.44-fold lower risk of starting smoking than those possessing the wild-type genotype of CYP2A6. In other words, the OR is 2.27-fold higher in subjects carrying the CYP2A6 wild-type/variant COMT rs4680 genotype than in those possessing the CYP2A6 low activity/variant COMT rs4680 genotype. The CYP2A6 and COMT genes were reported to be associated with smoking status. CYP2A6*4C is a whole-deletion type, decreased nicotine metabolism polymorphism [CYP2A6*4C gene was significantly higher among non-smokers than smokers [CYP2A6*4C genotype had a lower risk of smoking initiation and smoking persistence than those with the CYP2A6*1/CYP2A6*1 genotype [CYP2A6 in smokers appears to be associated with fewer cigarettes smoked, later initiation of smoking regularly, shorter smoking duration, and lower likelihood of smoking cessation [CYP2A6 slow inactivator genotype increased the risk of nicotine dependence when smoking was initiated during adolescence. However, it reduced the risk of smoking initiation, lowered cigarette consumption, and decreased the duration of smoking among adult dependent smokers [CYP2A6 slow inactivators, may affect tolerance and withdrawal mechanisms among these individuals [CYP2A6 slow inactivators smoke fewer cigarettes and tend to be less dependent on nicotine than smokers with normal activity alleles [CYP2A6 slow metabolism was associated with increased adolescent smoking cessation in Caucasian individuals [COMT rs4680 G/G  carriers than in those possessing the COMT rs4680 variant among healthy Caucasian men of Croatian origin [COMT rs4680 variation  was associated with nicotine dependence in men and women of African-American and European-American descent [CYP2A6 genotype and low-activity COMT genotype is associated with the risk of starting smoking.Our results show that the distribution of morphism . In Japa smokers , 35. Amogenotype . For peoessation . For Cau smokers . The conividuals . Smokers alleles . With re alleles . Very reividuals . The ORsn origin , American origin , and Japn origin . On the  descent , smoking descent , heavine descent , and sus descent . Our resCOMT genes than in those with high-activity COMT genes. Additionally, it was higher in subjects with the COMT rs4680 variant/COMT rs165599 variant than in those with the COMT rs4680 wild-type/COMT rs165599 variant. The urinary cotinine concentration is a reliable easy-to-use marker for plasma levels of cotinine and the sum of nicotine metabolites in smokers [CYP2A6 and COMT were associated with high nicotine dependence scores and heavier smoking, as we reported in this study. For Taiwanese university students, the primary reason for the first contact with smoking was curiosity. Anxiety, avoiding stress, and the difficulties of smoking cessation explained continuing smoking behavior among university students [CYP2A6 and COMT genes) among Taiwanese smokers in the university setting.We also found that the adjusted urinary cotinine concentration was higher in subjects with low-activity  smokers . Therefo smokers . This fistudents . The envThere are several limitations to our study. The percentage of female smokers in the Taiwanese population is low, only 4.4%, and they are difficult to recruit as participants. In our report, study participation was restricted to young male Taiwanese smokers. Although the total number of study subjects was relatively large, the numbers of individuals with some of the genotypes were too small to reach statistically significant power. Therefore, further studies with a larger sample size and that include female smokers are needed.COMT gene and the interaction between the CYP2A6 and COMT genes affect smoking status in young Taiwanese men. Effective smoking prevention and cessation intervention programs are required to reduce smoking among university students [CYP2A6 genotype and low-activity COMT genotype is associated with the risk of starting smoking. In addition, interaction of the DRD2 TaqIB and MAOA genes also affects smoking intensity in Taiwanese young men [A single nucleotide polymorphism rs4680) in the 80 in theoung men . This knoung men .Additional file 1: Table S1. Results of PCR–RFLP fragments for CYP2A6.Additional file 2: Table S2. Odds ratios for the effect of CYP2A6 and COMT.Additional file 3: Table S3. Comparisons of FTND, PCDS and CWS-21 among subjects carrying different CYP2A6 and COMT polymorphisms.


Copper-transporter ATP7B maintains copper homeostasis in the human cells and delivers copper to the biosynthetic pathways for incorporation into the newly synthesized copper-containing proteins. ATP7B is a target of several hundred mutations that lead to Wilson disease, a chronic copper toxicosis. ATP7B contains a chain of six cytosolic metal-binding domains (MBDs), the first four of which (MBD1-4) are believed to be regulatory, and the last two (MBD5-6) are required for enzyme activity. We report the NMR structure of MBD1, the last unsolved metal-binding domain of ATP7B. The structure reveals the disruptive mechanism of G85V mutation, one of the very few disease causing missense mutations in the MBD1-4 region of ATP7B. At the basal copper levels, ATP7B is located in the trans-Golgi network, where it delivers copper to the newly synthesized copper-containing proteins, such as ceruloplasmin. At the elevated copper levels, ATP7B relocates to the cytosolic membrane vesicles and plasma membrane, where it exports copper from the cell. This function is particularly important in the liver, where copper excretion into the bile serves as a main route of excess copper disposal in the human body.Membrane transporter ATP7B regulates the level and intracellular distribution of copper in human tissues7. There is no high-resolution structure of ATP7B, but the structures of most cytosolic domains have been solved by NMR, and the overall structure of ATP7B has been modeled by homology5 using the X-ray structure of the bacterial copper ATPase CopA from Legionella pneumophila2 as a template. This model does not include the N-terminal chain of the six cytosolic metal-binding domains (MBDs) connected by flexible loops of various length, a unique structural feature of ATP7B and of the closely related copper transporter ATP7A CxSCxxxIE motif responsible for binding copper(I) ions12 through the sulfur atoms of the two cysteine residues  values over 0.98 indicating a rigid local structure, with very limited backbone flexibility phosphine.MBD1 was expressed as fusion with the chitin-binding domain and intein using vector pTYB12 (New England BioLabs). The protein was isotopically labeled with 34 and analyzed using NMRView35. Backbone chemical shift assignments for MBD1 were made from HNCO, HNCA, HNCACB, HN(CA)CO, and HN(CO)CACB experiments36. Side-chain assignment experiments were done using H(CCO)NH, CC(O)NH, AND 1H, 15N-TOCSY. The backbone chemical shifts were assigned using CARA37. Hydrogen bond distance restraints were derived from H/D exchange measurements.NMR experiments were performed on the 600 MHz and 750 MHz Bruker NMR spectrometers equipped with a CryoProbe. Data were processed using NMRPipe1H, 15N-NOESY and 3D 1H, 13C-NOESY experiments, as well structure calculation was done using PONDEROSA38. Peak tables and distance restraint generation from 3D 1H, 15N-NOESY and 3D 1H, 13C-NOESY experiments, as well structure calculation was done using PONDEROSA-C/S38 with XPLOR-NIH based calculation options39. NOE cross peaks were automatically assigned with AUDANA40 and manually verified by PONDEROSA Analyzer coupled with NMRFAM-SPARKY41 and PyMOL (Schroedinger LLC). Final structure calculation step was carried out with explicit water refinement option with validated restraints that selects best 20 from 100 calculated models with the lowest energy criteria. The structure has been deposited in RCSB as entry 2N7Y.Peak tables and distance restraint generation from 3D 42 were performed essentially as described previously25, except that HSQC rather than TROSY versions of NMR experiments were used.NMR relaxation measurements, and data analysis using TENSOR 2.0Supplementary material


CDKN1A might act as a molecular switch to control spatial interactions between regulatory elements.A regulatory program involving hundreds of genes is coordinated by p53 to prevent carcinogenesis in response to stress. Given the importance of chromatin loops in gene regulation, we investigated whether DNA interactions participate in the p53 stress response. To shed light on this issue, we measured the binding dynamics of cohesin in response to stress. We reveal that cohesin is remodeled at specific loci during the stress response and that its binding within genes negatively correlates with transcription. At p53 target genes, stress-induced eviction of cohesin from gene bodies is concomitant to spatial reorganization of loci through the disruption of functional chromatin loops. These findings demonstrate that chromatin loops can be remodeled upon stress and contribute to the p53-driven stress response. Additionally, we also propose a mechanism whereby transcription-coupled eviction of cohesin from  It requires the establishment of an intricate gene expression program coordinated by the tumor suppressor transcription factor, p53. In response to stress, p53 integrates signals and directs cell fate by regulating the expression of genes involved in major cellular pathways fication , 2. Depee.g. enhancers, silencers) to their target promoters [Pioneering work recently revealed that the human genome has a spatial organization shaped by chromatin loops –8. The fromoters , 5, 15. romoters –18. ConsCDKN1A. Together, these findings demonstrate that chromatin loops are remodeled during the cellular stress response and contribute to the p53 transcriptional program.Given the importance of the spatial organization of the genome in the regulation of transcription, we explored whether DNA interactions are remodeled in response to stress to help establish the p53 transcriptional stress response program. To shed light on this issue, we used a genome-wide approach to investigate the binding of the cohesin subunit Rad21 in response to stress. Here, we show that cohesin is remodeled at specific loci during stress response. Notably, we unveil that cohesin is evicted from p53 target gene bodies during stress-induced transcription. Furthermore, using high-resolution Circular Chromosome Conformation Capture coupled to deep sequencing (4C-seq), we reveal that cohesin’s eviction from p53 target gene bodies in response to stress coincides with the disruption of chromatin loops that shape the spatial architecture of these loci. Finally, we show that these stress-dependent chromatin loops are important because they contribute to gene regulation of Daunorubicin and Polyethylenimine (PEI) were obtained from Sigma.2-containing atmosphere in DMEM (Wisent) supplemented with 10% fetal bovine serum (Sigma), 0.2U/mL penicillin G, and 100 mg/mL streptomycin (Invitrogen). Unless specified otherwise, cells stress treatment consisted of 250 nM daunorubicin (Sigma) for 8 h. All results presented with SEM were obtained from independent biological triplicates. Statistics were calculated using Student’s t-test.HCT116 p53+/+ and HCT116 p53-/- cells  were cultured in a 5% COChIP assays were performed as described previously using the antibodies listed in Rad21 ChIP-seq was performed similarly to regular ChIP experiments except for the following steps. For each treatment conditions six immuno-precipitations were performed. Immuno-precipitations were performed using Dynabeads coupled to protein A (Life Technologies) without pre-clearing. The libraries for deep-sequencing were prepared from 10 ng ChIPed DNA using the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB) according to manufacturer’s protocol. Samples were sequenced paired-end at 50-bp read length on an Illumina HiSeq-2000. See supplementary mRNAs of p21, p21C and 36B4 were quantified by RT-qPCR and normalized to 36B4 expression level. Total RNA was extracted from cultured cells using GenElute (Sigma) and reverse transcribed using the M-MLV reverse transcriptase enzyme (Enzymatics) and random hexamers according to manufacturer’s protocols. The RT-qPCR primer sets are reported in Total RNA was extracted from HCT116 p53+/+ cells using GenElute (Sigma) and treated with DNaseI (Ambion). The libraries for deep-sequencing were prepared from 800 ng total RNA using Illumina’s TruSeq Stranded mRNA LT Kit according to manufacturer’s protocol. Samples were sequenced paired-end at 50-bp read length on an Illumina HiSeq-2000. See supplementary Cells were grown in a 150 mm petri dish to 50–70% confluence and were then crosslinked in 1.1% formaldehyde for 10 min then washed two times in cold PBS. Cell pellet was resuspended in 500 μl of lysis buffer  and incubated for 30 min at 4°C on a rotary wheel. Subsequently, lysate was sonicated to obtain DNA fragments of 500 bp and was then centrifugated at 14,000 rpm for 10 min at 4°C. Supernatant was recovered and a 3 μl aliquote was collected as 1% input. Next, 500 μl of phenol/chloroform and 200 μl of lysis solution were added to 300 μl of lysate, the solution was then vortexed and centifugated at 12,000 rpm for 5 min. The aqueous phase was collected and transferred to a Gel-Lock tube containing 500 μl of phenol/chloroform. The solution was then mixed by inversion and centrifugated at 14,000 rpm for 1 min. The aqueous phase was collected, transferred to a tube containing 100 μg of proteinase K, and then incubated over-night at 65°C. Parallel to this, 500 μl of TE as well as 100 μg of proteinase K were added to the input aliquote, which was then incubated over-night at 65°C. The next day, 50 μg of RNase A were added to the samples, which were then incubated for 1 h at 37°C. DNA was then purified using QIAquick PCR Purification Kit and eluted using 700 μl of elution buffer. qPCR were subsequently performed using the primers reported in the 4C experiments were performed and analyzed as previously described , 21. ResWe used pGL3 basic, pGL3 promoter, and pRL-SV40 (Promega) to assess promoter, enhancer and repressor activity of putative regulatory elements. Cells were cultured in six-well plates, transfected using PEI with 100 ng of pGL3 vector, 2.5 ng of pRL-SV40, and 1000 ng of pGEX as carrier DNA, and collected 24h later. When a daunorubicin treatment was performed, HCT116 cells were transfected and treated 16 h later with 250 nM daunorubicin for 8h. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System kit from Promega and a Lumat LB 9507 luminometer from Berthold Technologies. Firefly signal was normalized to renilla signal. The sequences cloned in pGL3 vectors are reported in the 5’-ACACGACGrCrUrCrUrUrCrCrGrArUrCrU-3’ was then ligated to the mRNA. A 19 μl reaction containing 1 μg of purified RNA, 40 pmoles of 5’ adaptor, 1X T4 RNA ligase 1 buffer (New England Biolabs), 10% DMSO was assembled, incubated for 1 min at 65°C and then placed on ice. The reaction was then completed by adding 1 mM ATP, 20 u of RNasin (Promega), 30 u of T4 RNA ligase 1 (New England Biolabs) for a final volume of 25 μl and was incubated for 1 h at 37°C. RNA was then purified using SPRI agencourt RNAclean XP according to manufacturer’s instructions using a volume ratio of 1.8. A reverse transcription was then performed at 45°C using M-MLV reverse transcriptase enzyme (Enzymatics) according to manufacturer’s instructions using a gene specific primer 1 (GSP1)  and treated with DNaseI (Ambion). 1μg of total mRNA was then treated for 1 h with Tobacco Acid Pyrophosphatase (Epicentre) according to manufacturer's instructions and then purified using RNA Clean & Concentrator-5 (Zymo Research Corporation). The 5’ adaptor SP1) see . A PCR wimer see . PCR proet al. who showed that cohesin is evicted from the p53 target gene CDKN1A following 5-fluorouracil treatment, supporting that cohesin could be remodeled from its binding sites in response to stress [i.e. chromatin loops might be disrupted when cohesin is evicted from its binding sites. Hence, we used the cohesin subunit Rad21 as a proxy for cohesin and performed a ChIP-seq of Rad21 in HCT116 p53+/+ cells treated with the DNA damaging agent daunorubicin. RNA-seq experiments were carried out in parallel to obtain the transcription level of genes following stress induction. The biological replicates of these experiments exhibited a high degree of correlation  of Rad21 in HCT116 p53+/+ and HCT116 p53-/- cells treated or not with daunorubicin. We measured the binding of Rad21 at the CDKN1A (p21) site because CDKN1A induction in response to daunorubicin is virtually abolished in HCT116 p53-/- cells  [CDKN1A locus in response to stress: 1) the intragenic cohesin site, 2) the CDKN1A promoter, and 3) the NDR located downstream from the cohesin site  . Converste (TSS) –28. Nextte (TSS) . FAIRE rte (TSS) . We thussin site .CDKN1A locus in response to stress using high-resolution 4C   in HCT116 cells treated with daunorubicin. CDKN1A NDR and the 589–989 bp DNA fragment. We next investigated the nature of the transcript starting from this internal promoter. Nozell and Chen previously reported that p21C, a p21 variant, is transcribed from intron 1 of CDKN1A. p21C differs from p21 mRNA by its first non-coding exon that splices with the exon 2 of p21 [CDKN1A NDR, we speculated that the internal promoter might be responsible for the expression of p21C. Therefore, we determined the 3’ splice junction between the 5’ RACE product and the exon 2 of p21. A PCR using a forward primer overlapping the 5’ RACE product and a reverse primer located within exon 2 of p21 confirmed that the 5’ RACE product is spliced with exon 2 of p21  [Our data reveal that specific loci exhibit profound alteration in Rad21 binding following stress treatment. This happens equally at sites located outside genes or within introns and exons. However, we show that for Rad21 sites located within genes there is a correlation between the binding of Rad21 and the transcriptional status of the gene. For genes induced in response to stress, Rad21 binding decreases as gene expression increases, which is noticeable at p53 target genes . In the  cohesin . At leasg. Wapl) . Howeverg. Wapl) . Rather,g. Wapl) , 42. ThiCDKN1A and FDXR using high-resolution 4C reveals a correlation between cohesin eviction and the disruption of chromatin loops. We show that in non-stressed cells the CDKN1A locus is shaped by pre-established chromatin loops: a large interacting hub connects the CDKN1A promoter, PANDAR, and a cluster of three p53 binding sites, while the CDKN1A alternative internal promoter for p21C interacts solely with a downstream repressor element  Correlation between Rad21 ChIP-seq biological replicates. (B) Correlation between RNA-seq biological replicates. (C) Distribution of Rad21 ChIP-seq peaks with a Z-score <-2 and >2 within the genome. (D) RNA-seq and Rad21 ChIP-seq tracks obtained in HCT116 p53+/+ non-treated (NT) or treated with daunorubicin (Dauno) for the FDXR locus. (E) ChIP-seq signal of Rad21 binding sites surrounding the CDKN1A and FDXR genes. When Rad21 sites are located within genes, the transcription level fold induction of the gene following daunorubicin is indicated. (F) Correlation of Rad21 binding signal fold change with gene transcription fold change for Rad21 binding sites located within genes. (G) Correlation of Rad21 peak height and gene transcription FPKM for Rad21 binding sites located within genes. Data obtained in daunorubicin treated HCT116 cells. (H) p21 mRNA level assayed by RT-qPCR in HCT116 p53 +/+ and p53 -/- cells treated or not with daunorubicin.((DOC)Click here for additional data file.S2 FigA) mRNA induction in response to stress measured by RNA-seq for the p53 target genes FDXR, TP53i3, and GDF15 (B) ChIP of Rad21 and Smc1A at cohesin binding sites located within gene bodies of FDXR, TP53i3, and GDF15 in HCT116 p53+/+. (C) ChIP of Rad21 and Smc1A at cohesin binding sites located within gene bodies of FDXR, TP53i3, and GDF15 in HCT116 p53-/-.((DOC)Click here for additional data file.S3 FigA) The CTCF-Cohesin site present within the first intro of CDKN1A is conserved between different cell types. CTCF and Rad21 ChIP-seq signal at the CDKN1A gene is shown for different cell types. Representation adapted from the UCSC genome browser using tracks from the ENCODE project. (B) ChIP of CTCF and Rad21 performed in HCT116 p53+/+ cells. The signal obtained at the CDKN1A cohesin site is shown. (C) FAIRE experiment and ChIP experiment of H3K4me1, H3K4me3, and H3 carried out in non-treated HCT116 cells. H3K4me1 and H3K4me3 ChIP results were normalized to H3 to account for nucleosome density. (D) CDKN1A gene with the nucleosome depleted region shown in red. The DNaseI 125 cell types track , and the ChIP-seq track for Rad21 obtained in HCT116 p53+/+ non-treated are also shown. Adapted from the UCSC genome browser.((DOC)Click here for additional data file.S4 FigA) Representation of the CDKN1A gene with the promoter, the cohesin, and the NDR 4C viewpoints. 4C DpnII restriction sites are shown in red. The ENCODE DNaseI tracks  adapted from the UCSC genome browser is also shown. (B) Biological and technical replicate of the high-resolution 4C experiment carried out in HCT116 cells treated or not with daunorubicin using DpnII as first cutter. The NDR viewpoint track is shown. (C) High-resolution 4C experiment performed using Csp6I as first cutter (instead of DpnII) in HCT116 cells treated or not daunorubicin. The NDR viewpoint track is shown. (D) High-resolution 4C experiment carried out in HCT116 p53-/- cells treated or not with daunorubicin using DpnII as first cutter. The NDR viewpoint track is shown.((DOC)Click here for additional data file.S5 FigCDKN1A locus. HCT116 p53+/+ cells were treated (Dauno) or not (NT) with daunorubicin. The 4C tracks for the cohesin site and the interacting region are presented with their respective viewpoints in red. Rad21 ChIP-seq tracks are also shown. P53 binding sites are shown in green in the gene track.4C high-resolution interaction map of the (DOC)Click here for additional data file.S6 FigA) Luciferase reporter assay to test if the internal promoter activity is affected by stress. (B) Luciferase reporter assay to test if the activity of the internal promoter is p53-dependent. (C) PCR products obtained with primers amplifying the 5’RACE / p21 exon 2 junction using gDNA as template or cDNA from daunorubicin treated HCT116 cell.((DOC)Click here for additional data file.S7 FigA) 4C track of the NDR viewpoint obtained in non-treated HCT116 cells aligned to the ENCODE DNaseI track . Adapted from the UCSC genome browser. (B) FAIRE experiment carried out in non-treated HCT116 cells. Primer sets used were tailing the DNaseI peak located at chr6:36,660,086–36,660,295 strand (+) hg19. (C) Luciferase reporter assay to test the distal enhancer activity of the interacting region regulatory element. The signal is normalized to the Internal promoter/Luciferase construction. (D) Luciferase reporter assay to test the proximal enhancer activity of the interacting region regulatory element. The signal is normalized to the Control DNA1/Internal promoter/Luciferase construction. (E) Luciferase reporter assay to test the promoter activity of the interacting region regulatory element. The signal is normalized to the Control DNA1/Luciferase construction. (F) Luciferase reporter assay to test if the interacting region regulatory element represses SV40 enhancer activity. The signal is normalized to the SV40 promoter/Luciferase construction.((DOC)Click here for additional data file.S8 FigVenn diagram of the intersection of p53 binding sites and Rad21 binding sites in HCT116 p53+/+.(DOC)Click here for additional data file.S1 File(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S7 Table(DOC)Click here for additional data file.S8 Table(DOC)Click here for additional data file.S9 Table(DOC)Click here for additional data file.