Hence, the structural differences between individual E6 and their malleability appear, together with HPV type-specific surface exposed side-chains, to provide the structural basis for the different interaction networks reported for individual E6 proteins. Furthermore, the interaction of 51Z2 with a PDZ domain of hDlg was analyzed. Human Dlg constitutes a prototypic representative of the large family of PDZ proteins regulating cell polarity, which are common targets of high-risk HPV E6. Nine C-terminal residues of 51Z2 interact with the second PDZ domain of hDlg2. Surface plasmon resonance in conjunction with the NMR spectroscopy derived complex structure (PDB 2M3M) indicate that E6 residues N-terminal to the canonical PDZ-BM of E6 significantly contribute to this interaction and increase affinity. The structure of the complex reveals how residues outside of the classical PDZ-BM enhance the affinity of E6 towards PDZ domains. Such mechanism facilitates successful competition of E6 with cellular PDZ-binding proteins and may apply to PDZ-binding proteins of other viruses as well.The high-risk human papilloma virus (HPV) oncoproteins E6 and E7 interact with key cellular regulators and are etiological agents for tumorigenesis and tumor maintenance in cervical cancer and other malignant conditions. E6 induces degradation of the tumor suppressor p53, activates telomerase and deregulates cell polarity. Analysis of E6 derived from a number of high risk HPV finally yielded the first structure of a wild-type HPV E6 domain (PDB 2M3L) representing the second zinc-binding domain of HPV 51 E6 (termed 51Z2) determined by NMR spectroscopy. The 51Z2 structure provides clues about HPV-type specific structural differences between E6 proteins. The observed temperature sensitivity of the well-folded wild-type E6 domain implies a significant malleability of the oncoprotein  Infections are responsible for 17.8% of all cancers The HPV oncoproteins E6 and E7 of tumor-associated so-called “high-risk” HPVs prevent the differentiation of HPV-infected keratinocytes and immortalize primary, cultured human keratinocytes The HPV oncoprotein E6 has no known enzymatic activity. It interacts with numerous cellular proteins and these interactions contribute to reprogramming keratinocytes so as to prevent normal differentiation and to maintain cellular replication competence in order to amplify the HPV genome The inactivation of p53 is a general hallmark of tumorigenesis and tumors frequently show mutations and/or reduced p53 levels via induction of telomerase reverse transcriptase at the transcriptional level e.g. HPV 16, 18 and 51 A second common feature of tumors constitutes an increase in telomerase activity via its C-terminal PDZ-binding motif (PDZ-BM) in vivo mouse model resulted in smaller cervical tumors Furthermore, E6 of high-risk types interacts with a number of PDZ-domain containing proteins (PDZ proteins) in vivo is the multi-domain protein hDlg (human Dlg/hDlg1/SAP-97) in vitro degradation assay for a number of high-risk HPVs, among them HPV 16, 18 and 51 One PDZ domain containing protein targeted by E6 for degradation 29-CXXC Full-length E6 consists of approximately 150 residues and includes two zinc-binding domains (ZBDs) each coordinating one zinc ion via cysteines arranged in a motif of the form CXXC-XUp to now, no structural data on a wild-type HPV E6 are available. So we reasoned that addressing wild-type E6 proteins of high-risk types other than HPV 16 could on the one hand lead to a structure of a wild–type E6 and on the other hand shed light on structural similarities and differences among the E6 proteins including their interaction with target proteins. Here we determined the solution structure of the wild-type C-terminal zinc-binding domain of E6 derived from the high-risk HPV 51 and unraveled the structural basis of its interaction with the PDZ domain 2 of hDlg.6-tagged, recombinant E6 constructs derived from the high-risk types 16, 18, 26, 31, 45, 51, 59 and 97 as well as for the non-tumorigenic cutaneous type 1a was performed after bacterial expression in presence of 10 µM of Zn2+ as solubility of E6 constructs is dependent on low µM concentrations of zinc E. coli had been employed .Assessment of solubility of amino-terminally Hisemployed . The sol1H,15N]-HSQC spectra were recorded (see SI for details). If such a spectrum contained an acceptable signal distribution, homogeneous peak intensities and a signal number consistent with the expected number of observable amide groups, the respective protein was considered amenable to further NMR spectroscopic analysis. In the end, only 26Z2 and 51Z2 fulfilled these criteria . In the calculated structure, Ile88 Hδ2 is located directly above the aromatic ring of Trp132, while Ile88 Hδ1 is proximal to the aromatic ring of Phe125. Thus, ring current effects The calculated structural ensemble  exhibits10 helix. One anti-parallel β-sheet on 51Z2 is composed of the strands β1, β4 and β5, stabilized by charge-charge interactions and side chain hydrogen bonds, e.g. from R81 Hη12 to E127 Oε1. A second short anti-parallel β-sheet consisting of β2 and β3 is arranged almost perpendicular to the first sheet. This allows for the formation of hydrogen bonds between R102 HN and W132 O, H104 HN to G134 O and G134 HN to R102 O which stabilize the arrangement of both β-strands  of HPV 51 E6  and represents the first structural characterization of a non-mutated HPV E6 domain. The International Agency for Research on Cancer classifies HPV 51 as high-risk in vitro suggest protein motion at a timescale that causes line broadening at higher temperatures, whereas the zinc coordinating residues remain rigid. These findings derived from a wild-type E6 domain allow us to suggest that in cervical epithelial cells, where the E6 protein is expressed at 37°C, this particular domain and potentially the entire E6 protein may be more malleable in vivo than their published structures suggest at first glance. As the fold is re-adopted upon cooling down  E6 in complex with the LD1 motif of paxillin reveals an identical general topology for E6 . Thus, iE6 types  but rareE6 types . The coni.e. the target spectrum of closely related E6 is more similar than that of evolutionary more distantly related E6. Thus, although the E6 proteins may share a generally identical structural topology, subtle structural variations could explain the altered target spectrum of different E6 proteins Interestingly, a direct correlation between E6 phylogeny and their protein-protein interaction networks exists vide supra). In 51Z2 I128 is proximal to the conserved residues H126 and S82 on β4 and β1, respectively. HPV 51 is, as HPV 16, frequently detected in premalignant neoplasias, but proportionally significantly less present in cervical cancers e.g. E6AP or paxillin. Since the E3 ubiquitin ligase E6AP is often involved in E6-mediated target degradation Our analysis allows the identification of significant local structural differences between individual E6 proteins. Particularly, β4 and β5 of 51Z2 position differently as compared to the unbound ZBD2 of HPV 16 E6, but similar to the corresponding region of the only available full-length E6 structure in a complex, the BPV E6 bound to LD1 of paxillin . The convia contacts to bona fide PDZ domain residues [this study] or to residues outside the classical PDZ domain 51Z2 in the unbound state adopts a globular fold with a solvent exposed and flexible C-terminus  and S3 cversus E6CT6 and located far away from the peptide interface might have been perturbed due to the presence of such a long range network. This raises the question if the HPV 51 E6 extended PDZ-BM may even induce changes in places remote from the PDZ 2 of hDlg as the PDZ domains 1 and 2 of the hDlg-related PSD-95 are organized as a supramodule and retain their relative orientation in the context of the full-length protein A crystal structure of the C-terminal 11 residues of APC (APC_CT11) in complex with hDlgPDZ2 reveals only one peptide per asymmetric unit containing in turn five PDZ domains. Here, only the C-terminal six APC peptide residues gave rise to interpretable electron density in vivo with regard to interactions, such as e.g. with E6AP, may be addressed based on our findings in order to solve the puzzle of how the E6 proteins exert their malignant potential via interaction with a multitude of cellular targets.In summary, the first structure of a wild-type E6 domain and of the complex between its complete PDZ-BM and the PDZ domain 2 of hDlg revealed a number of E6 properties common for high-risk HPV E6 but also important differences between individual E6. The fully characterized interaction of E6’s extended PDZ-BM to one PDZ domain suggests future routes towards the elucidation of PDZ-supramodule E6 interaction. The implications of the E6 structural variations and the impact of the probable malleability of E6 E. coli BL21 (DE3). Cells were disrupted and protein solubility in cell-lysates was assessed. Soluble proteins were FPLC-purified and initially characterized by CD spectroscopy, dynamic light scattering, gel filtration chromatography and NMR spectroscopy. The affinity of E6-derived peptides to hDlgPDZ2 was elucidated utilizing Surface Plasmon Resonance. hDlgPDZ2 was generated by bacterial expression and FPLC purification. The generation of the labeled E6CT11 peptide was performed as previously described A detailed description of methods is provided in Figure S1Spectral changes of 26Z2 over time. Freshly prepared 15N-labeled 26Z2 (250 µM) was subjected to -HSQC NMR spectroscopy (blue con-tours). After 10 days at 4°C, the spectra (red contours) showed significant differences. Low peak-dispersion suggests an increased proportion of unfolded protein. Both spectra were recorded with 16 scans at a Bruker Avance III 750 MHz NMR spectrometer. Sample conditions were 135 mM NaCl, 45 mM L-Arg, 45 mM L-Glu, 9 mM DTT, pH 7.4, 4°C.(TIF)Click here for additional data file.Figure S2Biophysical characterization of 51Z2. For experimental details, see supplementary text 1. A Analytical gel-filtration. The chromatogram of 51Z2 run on a TSK gel G3000SWxl column is presented in blue, the column calibration is shown in orange with molecular weights of reference proteins indicated. 51Z2  eluted as a 9 to 10 kDa sized protein indicating a monomeric state. B Dynamic light scattering. 51Z2 exhibits a hydrodynamic radius of 1.58 nm, which corresponds to an approx. 10 kDa sized protein assuming a globular shape. C Circular dichroism spectrum of purified 51Z2. D The secondary structure content of 51Z2 was estimated from the CD spectrum C using CDNN (TIF)Click here for additional data file.Figure S3Sequence alignment of oncogenic E6 proteins. ClustalW2 alpha-papillomaviridae and to the species indicated on the left table-side.(TIF)Click here for additional data file.Figure S4Alignment of the high-risk HPV 51 E6 to representative low-risk and cutaneous E6 proteins. The residues corresponding to conserved positions among high-risk types Click here for additional data file.Table S1E6 domain architecture and solubility of recombinant E6 constructs.(PDF)Click here for additional data file.Table S2Final screening results of E6 constructs for NMR spectroscopy.(PDF)Click here for additional data file.Table S3Details of NMR experiments and samples for the 51Z2 structure determination.(PDF)Click here for additional data file.Table S4Details of NMR experiments and samples for the hDlgPDZ2-E6CT11 complex structure determination.(PDF)Click here for additional data file.Text S1Methods.(PDF)Click here for additional data file.


Dopamine, through D2 receptor (D2R), is the major regulator of lactotrope function in the anterior pituitary gland. Both D2R isoforms, long (D2L) and short (D2S), are expressed in lactotropes. Although both isoforms can transduce dopamine signal, they differ in the mechanism that leads to cell response. The administration of D2R agonists, such as cabergoline, is the main pharmacological treatment for prolactinomas, but resistance to these drugs exists, which has been associated with alterations in D2R expression. We previously reported that dopamine and cabergoline induce apoptosis of lactotropes in primary culture in an estrogen-dependent manner. In this study we used an in vivo model to confirm the permissive action of estradiol in the apoptosis of anterior pituitary cells induced by D2R agonists. Administration of cabergoline to female rats induced apoptosis, measured by Annexin-V staining, in anterior pituitary gland from estradiol-treated rats but not from ovariectomized rats. To evaluate the participation of D2R isoforms in the apoptosis induced by dopamine we used lactotrope-derived PR1 cells stably transfected with expression vectors encoding D2L or D2S receptors. In the presence of estradiol, dopamine induced apoptosis, determined by ELISA and TUNEL assay, only in PR1-D2S cells. To study the role of p38 MAPK in apoptosis induced by D2R activation, anterior pituitary cells from primary culture or PR1-D2S were incubated with an inhibitor of the p38 MAPK pathway (SB203850). SB203580 blocked the apoptotic effect of D2R activation in lactotropes from primary cultures and PR1-D2S cells. Dopamine also induced p38 MAPK phosphorylation, determined by western blot, in PR1-D2S cells and estradiol enhanced this effect. These data suggest that, in the presence of estradiol, D2R agonists induce apoptosis of lactotropes by their interaction with D2S receptors and that p38 MAPK is involved in this process. Dopamine (DA) is the predominant catecholaminergic neurotransmitter in the mammalian brain and is involved in a variety of functions such as locomotion, reinforcement, food intake, emotion and neuroendocrine secretion. In the anterior pituitary gland, DA inhibits prolactin (PRL) synthesis and release, as well as lactotrope proliferation D2R exists as two alternatively spliced isoforms, long (D2L) and short (D2S). D2L differs from D2S by the presence of additional 29 amino acid residues within the third intracellular loop. D2L and D2S can couple to different G inhibitory proteins Cabergoline (CAB), a D2R agonist, is the most effective compound for pharmacological treatment of prolactinomas D2R is coupled to distinct intracellular pathways including different MAPKs in vivo model.In the present work, we studied the role of D2R isoforms, D2L and D2S in the apoptosis of lactotropes induced by DA. We also investigated the participation of p38 MAPK in this action. We confirm the influence of E2 in the proapoptotic action of CAB on anterior pituitary cells in an Also, we show that DA induces apoptosis of lactotropes through D2S receptor activation in an E2-dependent manner and that p38 MAPK is involved in this action.All procedures complied with the Ethical Committee of the School of Medicine, University of Buenos Aires and the NIH Guide for the Care and Use of Laboratory Animals.Plus) , guinea pig rat prolactin antiserum , anti-guinea pig rhodamine-conjugated secondary antibody , fluorescein isothiocyanate (FITC) Annexin-V , anti-p38 MAPK and anti-phospho-p38 MAPK antibodies  and cabergoline .All drugs, media and supplements were obtained from Invitrogen , except Dulbecco's modified Eagle's medium (DMEM), bovine serum albumin (BSA), 17β-estradiol (E2), DA, normal horse serum and protease inhibitor cocktail , fetal bovine serum , SB203580 , gentamicin , Vectashield , all terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) reagents , Cell Death Detection (ELISAad libitum. The rats were ovariectomized (OVX) under ketamine  and xylazine  anesthesia. For primary cultures, the rats were euthanized by decapitation 15 days after ovariectomy and anterior pituitary glands were removed. For in vivo experiments, the rats were s.c. implanted in the skin of the back with silicone capsules containing 1 mg of 17β-estradiol (E2) or empty capsules (OVX) at the time of ovariectomy. The capsules  were prepared in the laboratory and were filled with 50 µl of E2 solution (20 mg/ml) diluted in ethanol. When ethanol was evaporated the capsules were sealed with a silicone adhesive. These capsules induce plasma levels of E2 similar to those of rats at proestrus Adult female Wistar rats were kept in controlled conditions of light (12 hour light-dark cycles) and temperature (20–25°C). Animals were fed standard lab chow and water 2+ and Mg2+ (KRBCMF), pH 7.4. Finally, the cells were dispersed in KRBCMF by extrusion through a Pasteur pipette. Dispersed cells were washed twice and resuspended in DMEM-S with 10% FCS. Cell dispersion yielded about 3–3.5×106 cells/gland. Cell viability as assessed by trypan blue exclusion was over 90%. The cells were seeded onto coverslides in 24-well tissue culture plates (1×105 cells/ml/well) and cultured in DMEM-S with 10% FCS for 24 h. Then the cells were cultured in fresh medium containing 17β-estradiol  for 24 h and in the same media without serum for a further 24 h. After this period, cells were incubated in the same media containing different drugs according to each experiment.A pool of anterior pituitary cells from 5–8 OVX rats was used for each primary culture. Anterior pituitary glands were washed several times with DMEM supplemented with 10 µl/ml MEM amino acids, 2 mM glutamine, 5.6 µg/ml amphotericin B, 25 µg/ml gentamicin (DMEM-S) and 3 mg/ml BSA. Then the glands were cut into small fragments and dispersed enzymatically by successive incubations in DMEM-S with BSA, containing 0.75% trypsin, deoxyribonuclease type I  and 10% fetal calf serum previously treated with 0.025% dextran-0.25% charcoal (FCS) to remove steroids. The cells were then washed with Krebs-Ringer bicarbonate buffer free of Ca5 cells/ml/well) for the TUNEL method, in 24-well culture plates (5×105 cell/ml/well) for ELISA or in 6-well plates (1×106 cells/ml/well) for protein extraction. Then, the cells were maintained for 24 h in DMEM/F-12 supplemented with human transferrine (100 µM), insulin (5 µM), putrescine (1 µM) and sodium selenite (30 nM) without serum (DMEM/F-12-SS). Finally, the cells were cultured with vehicle  or E2 (1 nM) in DMEM/F-12-SS for 48 h and incubated in the same media containing different drugs according to each experiment.The PR1 cell line was derived from a pituitary tumor of a Fischer-344 ovariectomized rat treated with E2 for 3 months in vivo experiments were dispersed with trypsin/DNase as previously described 2). The cells were incubated with 5 µl FITC Annexin-V and 10 µl propidium iodide  for 15 min in darkness. Cells were immediately analyzed by flow cytometry  and data analyzed by WinMDI98 software. Annexin-V-positive/PI-negative cells were considered early apoptotic cells, whereas Annexin-V-negative/PI-positive cells were considered necrotic cells. Double-positive (Annexin-V-positive/PI-positive) cells were considered to be in a late stage of apoptosis.Anterior pituitary glands from Plus). This kit is used for relative quantification of histone-complexed DNA fragments (mono- and oligonucleosomes) induced by apoptosis. After treatment, cells were lysed to obtain the nucleosomes and processed according to the manufacturer's protocol. Optic density (OD) was measured in a spectrophotometer at 405 nm.The apoptotic response of PR1 cells was measured by Cell Death Detection  according to the manufacturer's protocol. After incubation with 10% horse serum, slides were incubated for 1 h with antidigoxigenin-fluorescein antibody  to detect incorporation of nucleotides into the 3-OH end of damaged DNA. Slides were mounted with Vectashield  containing 4,6 diamidino-2-phenylindoledihydrocloride (DAPI) for DNA staining and visualized in a fluorescence light microscope . In primary culture of anterior pituitary cells, lactotropes were identified by immunofluorescence. After incubation with 10% serum in PBS, the cells were incubated for 1 h with guinea pig rat prolactin antiserum (1∶1.500). Then, the slides were incubated for 1 h with aDIG (1∶10) and a rhodamine-conjugated anti-guinea pig secondary antibody (1∶200). The percentage of total apoptotic cells was calculated as ×100. The percentage of apoptotic lactotropes was calculated as (TUNEL-positive prolactin-immunoreactive cells/prolactin-immunoreactive cells)×100. The percentage of lactotropes determined by immunofluorescence was between 25–40% of total anterior pituitary cells.Total proteins were extracted from cultured cells in lysis buffer containing protease inhibitor cocktail and phosphatase inhibitors . The protein concentration of each sample was determined by Bradford protein assay . Twenty µg of total proteins were size-fractionated in 12% SDS-polyacrylamide gel and then electrotransferred to polyvinyl difluoride (PVDF) membranes. Blots were blocked for 2 h in 5% nonfat dry milk-TBS-0.1% Tween 20 at room temperature and incubated overnight with the appropriate primary antibody in the same buffer at 4°C. The primary antibodies used were: anti-p38 MAPK (1∶1000) and anti-phospho-p38 MAPK (1∶2000). Then, membranes were incubated for 1 h at room temperature with the corresponding peroxidase conjugated secondary antibody. Immunoreactivity was detected by enhanced chemiluminescence. Membranes were exposed to X-ray films and developed using an X-Ray developer.2 test Each experiment was performed at least twice. The percentage of apoptotic cells determined by flow cytometry was expressed as mean ± SE and evaluated by two-way ANOVA followed by Tuckey's test. The optic density measured by ELISA was expressed as mean ± SE and analyzed by one-way ANOVA followed by Dunnett's test. The number of apoptotic cells identified by TUNEL was determined in duplicate slides from independent experiments. Results were expressed as the percentage ±95% confidence interval (CI) of apoptotic cells of the total number of cells counted in each specific condition. Differences between proportions were analyzed by χin vivo model, apoptosis in the anterior pituitary gland was evaluated in OVX and E2-treated rats injected with CAB (determined by FICT Annexin-V staining). As previously described To confirm the permissive action of E2 on the apoptotic action of D2R activation in an To study the role of D2R isoforms in the apoptosis of lactotropes induced by DA, we evaluated the proapoptotic action of this catecholamine on PR1 cells expressing either D2L or D2S. Considering that DA-induced apoptosis of lactotropes is E2-dependent To investigate whether p38 MAPK is involved in the apoptosis of anterior pituitary cells induced by D2R activation, anterior pituitary cells from OVX rats cultured with E2, were incubated with DA or CAB in the presence of SB203580, a p38 MAPK inhibitor. SB203580 blocked the apoptotic effect of either DA or CAB in anterior pituitary cells and lactotropes from primary cultures . Also, iDA is the main inhibitor of lactotrope function and its interaction with D2R not only inhibits PRL secretion, PRL gene expression and lactotrope proliferation Since p38 MAPK has been implicated in DA-induced apoptosis in neurons in vitro and in vivo rodent models The first choice of treatment for prolactinomas are DA receptor agonists, being CAB the most effective one which inhibits PRL secretion and reduces tumor size E2 increases the expression of D2L mRNA in anterior pituitary cells Prolactinomas are the most frequent pituitary tumors and medical therapy with D2R agonists is highly effective in most cases. However, some patients do not respond to pharmacological treatment Our study shows that the apoptosis of lactotropes induced by DA is mediated by D2S and is an estrogen-dependent process, involving p38 MAPK activation. These observations suggest that a decrease in D2S expression may participate in D2R agonist resistant prolactinomas.


Artemisia annua L. AaPYL9 is expressed highly in leaf and flower. AaPYL9 protein can be localized in both nucleus and cytoplasm. Yeast two-hybrid assay shows AaPYL9 can specifically interact with AtABI1 but not with AtABI2, AtHAB1 or AtHAB2. ABA can enhance the interaction between AaPYL9 and AtABI1 while AaPYL9-89 Pro→Ser and AaPYL9-116 His→Ala point mutations abolishes the interaction. BiFC assay shows that AaPYL9 interacts with AtABI1 in nucleus in planta. Transgenic Arabidopsis plants over-expressing AaPYL9 are more sensitive to ABA in the seed germination and primary root growth than wild type. Consistent with this, ABA report genes have higher expression in AaPYL9 overexpressing plants compared to wild type after ABA treatment. Moreover, overexpression of AaPYL9 in A. annua increases not only drought tolerance, but also artemisinin content after ABA treatment, with significant enhancement of the expression of key genes in artemisinin biosynthesis. This study provides a way to develop A. annua with high-yielding artemisinin and high drought resistance.The phytohormone abscisic acid (ABA) plays an important role in plant development and environmental stress response. In this study, we cloned an ABA receptor orthologue, AaPYL9, from  ArabidopsisArabidopsis, the PYR1/PYL/RCAR family have 14 members (PYR1 and PYL1-13), which previously were classified as plant pathogenesis-related proteins of class 10 (PR-10) RD29 and RAB18 are induced ArabidopsisThe phytohormone abscisic acid (ABA) is a key regulator, involved in different plant developmental processes such as seed germination, root elongation, and bud dormancy Artemisia annua L. (qinghao), is a sesquiterpene lactone endoperoxide that provides the basis for effective treatments of malaria, especially for the cerebral and the chloroquine-resistant forms of this disease A. annua plant is still the only commercial source of artemisinin. Unfortunately, only very low amounts of artemisinin can be isolated from the aerial parts of A. annua (between 0.01 and 0.6% dry weight) Malaria is a global health problem with more than one billion people living in areas with a high risk of the disease Arabidopsis, secondary metabolic processes are also enriched in both guard cell and leaf ABA-induced genes Artemisia will provide the basis for artimisinin genetic engineering. Moreover, the enhancement of ABA sensitivity of plants has been proved to be an efficient way to increase biomass under abiotic stress, including drought A. annua has been cloned and functionally characterized.Interestingly, the content of artemisinin is induced by exogenous ABA A. annua and functionally studied in A. annua as well as in model plant Arabidopsis. AaPYL9 codes for a PYR/PYL/RCAR family protein, with high similarity to Arabidopsis PYL9. Yeast two-hybrid and BiFC assays show that AaPYL9 interacts with AtABI1 in yeast and in planta, while AaPYL9-86 Pro→Ser and AaPYL9-116 His→Ala point mutations abolishes the interaction. Overexpression of AaPYL9 (AaPYL9 OX) in Arabidopsis greatly reduced the seed germination rate under various abiotic stress treatments . Moreover, the AaPYL9 OX Arabidopsis showed reduced root elongation under ABA treatment during seedling growth. Further expression analysis showed that overexpression of AaPYL9 in Arabidopsis increased the ABA sensitivity in early seedling growth stage. Furthermore, overexpression AaPYL9 in Artemisia increased drought tolerance and improved artemisinin content. The cloning and functional analysis of A. annua ABA receptors provides a basis for further studying the roles of the ABA receptor on the regulation of artemisinin biosynthesis in A. annua plant.In the present study, we report that an ABA receptor, AaPYL9, was successfully cloned from A. annua was constructed using leaf as the source of mRNA. In total, 2,000 randomly picked clones were sequenced. By blast searching the 2,000 sequences, AaPYL9 was identified that encoded PYR/PYL/RCAR family member  while four PP2C-A protein, AtABI1, AtABI2, AtHAB1 and AtHAB2, were fused to the Gal4 DNA binding domain (BD domain). Only AtABI1 showed interaction with AaPYL9 and adding ABA can increase the interaction  and the fusion protein was driven under the control of the 35S promoter of Cauliflower mosaic virus (CaMV). ytoplasm , as expec) in the pEG201-YC vector. Moreover, AtABI1 was translationally fused to the N-terminal region of the yellow fluorescent protein (YFPN) in the pEG202-YN vector. The constructs were co-transfected into Arabidopsis protoplasts cells, and as a result, fluorescence was observed in the nucleus of protoplasts cells (The protein interaction between AaPYL9 and the AtABI1 proteins was further confirmed in plant cells by bimolecular fluorescence complementation (BiFC). AaPYL9 was translationally fused to the C-terminal region of the yellow fluorescent protein (YFPts cells . These dArabidopsis ABI1 prompted us to test whether overexpression of AaPYL9 in Arabidopsis would affect ABA sensitivity in seeds and early seedling growth. The intact AaPYL9 gene or with a point mutation (AaPYL9-H116A) were constructed under the control of 35S promoter and then introduced into Arabidopsis plants. Twenty transgenic lines of each construct were obtained and three T3 homozygous lines were chosen to be analyzed. Expression analysis showed that AaPYL9 gene and AaPYL9-H116A were strongly expressed in 35S::AaPYL9 and 35S::AaPYL9-H116A transgenic Arabidopsis plants  in the expression of RAB18, P5CS1, RD29A, and RD29B upon ABA treatment from that detected in wild-type plants . However, when we analyzed ABA treated leaves, the artemisinin content of wild-type Artemisia was increased by 33%, while the artemisinin content of 35S::AaPYL9 transgenic lines was increased by 74%∼95% , and the latch (H116A), both of them weakened the interaction with AtABI1 in yeast two-hybrid assays. Furthermore, we test the function of AaPYL9H116A mutant in planta, the seed germination and root elongation of 35S::AaPYL9-H116A were similar with wild-type. These results demonstrate that the AaPYL9 gate residue P89 and latch residue H115 are critical for interaction with AtABI1 and downstream signaling. Together, the ABA receptor of Artemisia has similar structural basis for ABA signaling with Arabidopsis, providing useful information for the evolution of ABA signaling perception and transduction.The discovery of PYR/PYL/RCAR proteins has led to a series of crystallographic studies AaPYL9 in Arabidopsis increased the plants' sensitivity to ABA and thus reduces the seedling growth under ABA, osmotic and salt stress treatments. In agreement well with this, transgenic Artemisia plants overexpressing AaPYL9 also shows drought tolerant phenotype, which may be of great value to the growth of this important medicinal plant. ABA affects the biosynthesis of several secondary metabolites, such as that of terpenoid indole alkaloids in Catharanthus roseusArabidopsisArtemisia plants before harvesting AaPYL9-OX Artemisia compared to wild type. The results demonstrated that overexpression of AaPYL9 caused increased artemisinin accumulation when treated with ABA. Although showing similar artemisinin content under normal growth condition, AaPYL9-OX plants had significantly more increase (∼1.7 fold) of artemisinin content after ABA treatment than wild-type (∼1.3 fold). The more increase of artemisinin production may be due to the more receptors in the transgenic plants which sense the exogenous ABA. Under normal growth condition, the ABA receptors in wide type may be excess and in the rest form, and the ABA concentration was quite low, thus overexpression of ABA receptor will not increase the sensitivity to the endogenous ABA. But upon ABA treatment, ABA levels increased to achieve marked phenotype. Overexpression of AaPYL9 in Arabidopsis, improved ABA signaling in the plant under ABA treatment, which can be revealed by expression of ABA responsive genes, such as P5CS1, RD29A, RD29B, RAB18 , the other chemicals were purchased from China National Medicines Corporation Ltd (http://www.sinopharm.com/).(+-)Abscisic acid was obtained from Sigma-Aldrich  or NaCl solution (200 µM) for 1, 3 and 6 h. For drought stress, 15-day-old A. annua were transferred from water-saturated soil to filter paper for 1, 3 and 6 hours. The Arabidopsis  wild-type and transgenic plants used in this work were Col-0 ecotype, and they were grown in pots in a growth chamber under 24°C and 16-h-light/8-h-dark photoperiod at 80 to 100 mE m−2 s−2. For in vitro culture, seeds were surface sterilized in 10% sodium hypochlorite solution and 0.01% Triton X-100 for 5 min, and washed three times in sterile distilled water. After conducted during 3 d at 4°C, the seeds were sown on plates containing MS solid medium composed of MS basal salts and 20% sucrose solidified with 0.6% agar with pH 5.7. Plates were sealed and incubated in a controlled-environment growth chamber. Seedlings of the A. annua were obtained from 4 weeks-stratified seeds sown in a controlled environment chamber under the same condition of Arabidopsis.Artemisia grown in greenhouse. Randomly picked clones were sequenced with an ABI 3700 DNA Sequencer, and sequences were assembled and edited using PHRED/PHRAP. The non-redundant unigenes (contigs and singlets) were subjected to similarity searches against the GenBank non-redundant (nr) protein databases using the BLASTX algorithm with default parameters, and then the AaPYL9/RACR1 sequence was identified.A cDNA library constructed in the vector using poly (A+) RNA from leaves of 4-month-old Point mutation of AaPYL9 was performed using overlapping PCR strategy. To make P89S mutation, overlapped fragments of AaPYL9 were amplified using KOD-plus DNA polymerase (TOYOBO) using two pairs of primers: AaPYL9-F and AaPYL9-P89S-R, AaPYL9-P89S-R and AaPYL9-R. The PCR products were separated by 1.2% agarose gel and the DNA was extracted using a Gel extraction kit (Invitrogen). Purified PCR products were mixed, and 1 µl of the mixed DNA was used as the template in PCR amplification by KOD-plus DNA polymerase using AaPYL9-F and AaPYL9-R primers for Y2H and overexpression vector construction. The same method was used to make H116A mutation. All the mutations were checked before use.-AaPYL9 was transferred to the bait vector pDEST32 by Gateway LR recombination reaction (Invitrogen). ABI1, ABI2, HAB1 and HAB2 from Arabidopsis were cloned into pENTR vector and then transferred to the split vector pDEST22. To prepare vectors for AaPYL9 localization, the plasmid pEarleyGate104-35S::YFP::AaPYL9 was generated by LR recombination reaction (Invitrogen). To overexpressing AaPYL9 in Arabidopsis we transferred the pENTR-AaPYL9 to the pEarleyGate101-35S::AaPYL9. The similar method was used to make AaPYL9-P89S and AaPYL9-H116A overexpression and Y2H vectors. BiFC vectors pEarleyagte201-YN and pEarleygate202-YC were kindly provided by Professor Steven J. Rothstein. pENTR-AaPYL9 was transferred into pEarleyagte202-YN and pENTR-AtABI1 was transferred into pEarleyagte201-YC by Gateway LR recombination reaction (Invitrogen), which were named pEG202yc-AaPYL9 and pEG201yn-AtABI1. pEG101-AaPYL9, pEG101-AaPYL9-P89S and pEG101-AaPYL9-H116A constructs were introduced into Arabidopsis using a flower dipping method EcoRI and BamHI ends, and inserted into the binary vector PHB under the control of the double CaMV 35S promoter. The resulting construct (PHB-AaPYL9) was introduced into Agrobacterium tumefaciens strain EHA105. A. annua transgenic plants were generated as previously described A. annua were surface-sterilized by treatment with 75% (v/v) ethanol for 1 min, followed by treatment with 20% (v/v) NaOCl (sodium hypochlorite) containing Tween-20 for 20 min, and washed three to four times with distilled water and finally sown in germination medium MS0 basal medium  with a photoperiod of 16 h light/8 h dark and light at 8000 lux  at 25°C. After two weeks, the germinated seedlings were collected and the leaves were cut into 0.5-cm-diameter discs and used as the explants in Agrobacterium-mediated co-cultivation. After hygromycin selection in the selective medium MS1 , the hygromycin-resistant plantlets were regenerated and then transferred into rooting medium MS2 . After one month, the rooted plantlets were transferred into soil into pots in the growth chamber.To generate the constructs, the open reading fragment of the AaPYL9 cDNA was amplified by PCR and cloned into pENTR vector (Invitrogen) and sequenced. For yeast two-hybrid screening, pENTRYFP::AaPYL9 was transferred into Arabidopsis protoplasts. Preparation and analysis of Arabidopsis protoplasts was performed as described by Moes et al. The expression vector pEarleyGate 104-35S::AaPYL9 was cloned in a bait vector pDEST32, and at the same time ABI1, ABI2, HAB1 and HAB2 from Arabidopsis were cloned in a split vector pEDST22. We co-transformed the specific pairs of BD and AD constructs into the yeast strain AH109 by the lithium acetate method, and the transformants were spread on media plates that lacked leu, trp (SD/-T-L). Five independent 3-d-old colonies with the same size were picked up and diluted into 100 µl 1×TE, and aliquots of 10 µl yeast cells were dropped on selective medium that lacked trp, leu, his(SD/-T-L-H) and lacked trp leu, his, ade(SD/-T-L-H-A) supplemented with or without 10 µM ABA.Arabidopsis protoplasts were transfected with plasmid pEG202yc-AaPYL9 and pEG201yn-AtABI1 DNAs (10–20 µg). YFP fluorescence was evaluated using a Fluoview FV300 CLSM system (Olympus Ltd) 3 h after transfection.For BiFC assay, Arabidopsis lines  and 35S::AaPYL9-H116A transgenic Arabidopsis lines  as well as ABA receptor triple mutant pyr1/pyl1/pyl4 Arabidopsis were sterilized and plated on solid medium composed of MS basal salts, 2% Glc, and different concentrations of ABA (0.3 and 0.5 µM). After 4 days of stratification at 4°C in the dark, plates were incubated at 22°C under 16-h light/8-h dark cycle. All seeds were harvested on the same day in identical environmental conditions. In order to score seeds germination, the seeds that formed green shoots were considered to be germination. The percentage of seeds was determined in all assays after 8 days of sown. Photographs were taken 8 days after sown. Each value represents the average germination percentage of approximately 200 seeds with the ±SE value of three replicates.To measure ABA sensitivity, approximately 200 seeds each from wild-type (Col-0), three independent 35S::AaPYL9 transgenic Arabidopsis lines  and 35S::AaPYL9-H116A transgenic Arabidopsis lines  as well as ABA receptor triple mutant pyr1/pyl1/pyl4 Arabidopsis were sterilized and plated on the MS medium and MS medium supplemented with 200 mM mannitol or 100 mM NaCl, respectively. Seedling establishment was scored as the percentage of seeds that developed green expanded cotyledons after 7 days of sowing. Data are means ±SE from three independent experiments (n≈200 seeds per experiment).To determine sensitivity to inhibition of germination by low osmoticum and salt, Approximately 200 seeds each from wild-type (Col-0), three independent 35S::AaPYL9 transgenic http://rsb.info.nih.gov/nih-image/). Data are averages ±SE from three independent experiments.To measure ABA hypersensitive inhibition of root growth, approximately 20 seedlings per experiment were sterilized and plated on MS medium for 4 days. Then, plants were transferred a new plate containing MS medium supplemented with or without 5 µM ABA. Photographs were taken after 7 d of transferring, and quantitatively analyzed using NIH IMAGE J software -PCR was extracted using RNA Extract Kit as described by the manufacturer (Tiangen). Real-time PCR was performed in a Bio Rad MJ (Bio Rad). Amplification was carried out with Brilliant SYBR Green QPCR MasterMix (Takara) according to the manufacturer's instructions. The thermal profile for SYBR Green real-time PCR was 95°C for 2 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The expression of all genes was normalized against the expression of the endogenous control gene (β-ACTIN8). All experiments were repeated at least three times.For analysis of ABA-responsive gene expression in transgenic Artemisia leaves were incubated for 2 h in a solution containing 10 mM KCl, 0.2 mM CaCl2, and 10 mM MES-KOH (pH 6.15) under white light for stomata fully open. Then, they were incubated for 2 h in the same buffer supplement or not with 10 µM ABA. Stomatal apertures were measured from images obtained by using Nikon ECLIPSE TS100 with IMAGEJ software. Each value represents the average aperture approximately 40 stomata with the ±SE value of three replicates.For the transpiration assay, the loss of fresh weight of approximate 1 g leaves at the same developmental stage was measured at room temperature after the indicated periods of time. To score ABA-induced stomatal closing, A. annua were sprayed by 10 µM ABA, and collected immediately which were defined as time 0, and leaves collected after 24 h were defined as time 24. The method of quantification of artemisinin was performed as previously described Leaves of Figure S1Expression analysis of AaPYL9 in different tissues and under ABA, NaCl and drought treatment. A, Expression analyses of AaPYL9 in different Artemisia annua L. tissues. Real-time PCR analysis of total RNA isolated from Artemisa annual L. untreated tissues. Quantitative RT-PCR was performed for the organ specific expression analysis and the internal control gene ACTIN was used to show the normalization of the amount of templates in PCR. B, Real-time PCR analysis of AaPYL9 expression in Artemisia treated with 10 µM ABA (gray bar) or treated with mock (black bar). C, Real-time PCR analysis of AaPYL9 expression in Artemisia treated with 200 µM NaCl (gray bar) or treated with mock (black bar). D, Real-time PCR analysis of AaPYL9 expression in Artemisia under drought condition. Students' t test showed that the expression of AaPYL9 have no significant change when treated with 10 µM ABA, 200 µM NaCl, as well as under drought condition. Data represent means ±SE from three replicates.(TIF)Click here for additional data file.Figure S2Yeast two-hybrid assay for AaPYL9- interacting proteins. Yeast two-hybrid interaction test of AaPYL9 with AtABI2, AtHAB1 and AtHAB2. AaPYL9 could not interact with AtABI2, AtHAB1, AtHAB2 in selective medium .(TIF)Click here for additional data file.Figure S3PCR analyses of independent transgenic A. annua plants. PCR analysis for the presence of AaPYL9 overexpression constructs in A. annua plants. WT: wild-type A. annua plant (negative control); P: Positive control (PHB-AaPYL9 plasmid). The forward primer used in this PCR analysis was located in 35S promoter, while reverse primer was located in AaPYL9 open reading fragment.(TIF)Click here for additional data file.Figure S4Stomata of the AaPYL9 overexpression Artemisia showed increased ABA response. A: photographs of stomatal of 35S::AaPYL9 transgenic Artemisia (9) and wild-type plants in the presence or absence of ABA. B: Average stomatal aperture of AaPYL9 overexpression transgenic Artemisia and wild-type plants in the presence or absence of ABA. Stomata aperture of the AaPYL9-9 plants significantly decreased compared with wild type. *P≤0.01 (student's t test). Data are averages ± SE from three independent experiments (n≈40 stomata per experiment).(TIF)Click here for additional data file.


GJB2 (CX26) and GJB6 (CX30) mutations are implicated in nearly half of nonsyndromic hearing loss cases. A recent study identified a heterozygous mutation, c.119C>T (p.A40V), in the GJB6 gene of patients with nonsyndromic hearing loss. However, the functional role of the mutation in hearing loss remains unclear. In this study, analyses of cell biology indicated that a p.A40V missense mutation of CX30 causes CX30 protein accumulation in the Golgi body rather than in the cytoplasmic membrane. The tet-on protein expression system was used for further study of mutant proteins in CX30 and CX30A40V co-expressions and in CX26 and CX30A40V co-expressions. The p.A40V missense mutation exerted a dominant negative effect on both normal CX30 and CX26, which impaired gap junction formation. Moreover, computer-assisted modeling suggested that this p.A40V mutation affects the intra molecular interaction in the hydrophobic core of Trp44, which significantly alters the efficiency of gap junction formation. These findings suggest that the p.A40V mutation in CX30 causes autosomal-dominant nonsyndromic hearing loss. These data provide a novel molecular explanation for the role of GJB6 in hearing loss.Dysfunctional gap junctions caused by  Hearing loss is a common sensory disorder in humans + ions, main charge carrier for sensory transduction + recirculation The inner ear consists of the hearing organ, the cochlea, are essential for hearing in the mammalian inner ears. The function of cochlea is dependent on tightly controlled ions, particularly KGap junction [GJ] channels mediate direct cell-to-cell communication by enabling intracellular transport of small biological molecules (<1 kDa), including electrolytes, second messengers and metabolites via gap junctions is crucial for auditory function. The important role of intercellular communication, particularly that between GJB2 and GJB6 (encoding CX26 and CX30), has been confirmed by evidence that certain connexin gene mutations cause sensorineural hearing loss GJB6 gene (AJ005585) encodes a 261-amino acid protein. The human CX30 protein shares 93% homology with mouse Cx30 and 76% identity with human CX26. Northern blot, RT-PCR, and in situ hybridization analyses have revealed mouse Gjb6 expression in the trachea, thyroid, brain, and cochlea GJB6 mutations, including DFNB1 and DFNA3, reportedly cause hearing loss GJB6 gene remains unclear. To elucidate the pathogenic role of CX30 variant in nonsyndromic hearing loss, the functional properties of variant CX30 gap junctions must be clarified. This study therefore investigated the effect of the p.A40V (c.119C>T) variant on functional properties and on the subcellular localization of the heterozygous variant CX30 protein in HeLa cells and in tet-on HeLa cells.Intercellular communication GJB6 were obtained from the genomic DNA using the PCR reaction. PCR was carried out with the following oligonucleotide primers: forward primer was GJB6 F-HindIII aagcttatggattgggggacgctg-3′5′- atg and corresponded to nucleotides 1–18 of the human GJB6 coding region; reverse primer was GJB6 R-Xho1 ctcgaggcgcttgggaaacctgtgattg -3′5′- atg and corresponded to nucleotides 762–783 of the human GJB6 coding region. The PCR DNA product (801 bp) of the human GJB6 coding region without stop coden was cloned into the pcDNA3.1-CT vector. Mutant CX30 gap junction proteins were obtained by performing oligonucleotide-directed mutagenesis using the Stratagene Quickchange site-directed mutagenesis kit . The following oligonucleotide primers (mutated nucleotide is underlined) were used to prepare the mutant CX30A40V gene: CX30A40V sense tccaggaagtgtggg -3′5′- ctagtggtggctg; CX30A40V antisense acagccaccactag -3′5′- cccacacttcctgg. For fusion protein generation, cDNA sequences of the autofluorescent reporter proteins EGFP  and DsRed  were fused in-frame to the C-terminus of WT and mutant CX30A40V. The open reading frames (ORFs) of GJB6 were obtained from the pcDNA3.1-CT clone after digestion with HindIII and SacII, and then subcloned into the HindIII and SacII restriction sites in vectors pEGFPN1 and pDsRedN1 . Further, the coding region of CX30WT and that of mutant CX30A40V were amplified from plasmids containing the CX30 cDNA (CX30WT-EGFP or CX30A40V-DsRed) using two pair primers containing recognition sequences 5′- SalI (gtcgactgatcagttatctagatccggatg and 3′- NotI (gcggccgcgctagcgctaccggactcatgNheI (agatccgctagcgctaccg) and 3′-EcoRV (gatatcccgctacaggaacaggtggatg), respectively, and Platinum Pfx DNA polymerase . Purified products were subcloned into the corresponding site of the bi-directional expression vector pBI . The dideoxy DNA sequencing method, using a DNA Sequencing Kit , an ABI Prism 3730 Genetic Analyzer , and restriction digest were used to confirm the DNA sequence of all constructs.) or 5′-The mammalian expression vector pcDNA3.1-CT used in this study was constructed as previously described GJIC gene were used throughout this study. Cell lines were maintained in standard cell culture medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 50 units/mL penicillin–streptomycin. Cell cultures were maintained at 37°C in a humidified 5% CO2 incubator. The vectors pcDNA3.1-CT, pGFPN1, and pDsRedN1 containing the DNA fragment encoding the wild-type or mutant CX30 protein were transfected to HeLa cells using lipofectAMINE . The post-transfection cell line was used for the subsequent functional analyses.Human epitheloid cervix carcinoma cells  lacking the GJIC gene was purchased from BD Biosciences Clontech  and maintained in Dulbecco's modified Eagle's medium, supplemented with 10% FBS , 100 m“Insert>Symbol”g/ml G418, 100 U/ml penicillin, and 100 m“Insert>Symbol”g/ml streptomycin at 37°C in a moist atmosphere containing 5% CO2. Transfection was carried out using LipofectAMINE reagent  according to the manufacturer's instructions. A ratio of 1 m“Insert>Symbol”g DNA vs. 2 m“Insert>Symbol”l LipofectAMINE 2000 was used for the tet-on HeLa cells. Cells were harvested at 24 h post-transfection and grown on a coverslip for 24 h at 37°C in a humidified 5% CO2 incubator. Then, tet-on HeLa cells were treated with 1 m“Insert>Symbol”g/ml doxycyclin (Dox)  in cell culture medium to induce CX26WT, CX30WT, CX30A40V or both WT and mutant protein expression.The tet-on HeLa cell line deficient in the Wild-type or mutant CX protein expression in HeLa and tet-on HeLa cells was analyzed by a direct fluorescent protein fusion method involving fusion of EGFP or DsRed to the C-terminal ends of the CX proteins. Briefly, post-transfection HeLa cells and tet-on HeLa cells grown on coverslips were fixed with 4% paraformaldehyde in 0.1 M PBS for 20 min and then rinsed three times in PBS. The tet-on HeLa cells grown on coverslips were exposed to Dox for 5 h prior to fixation. Then, the coverslips were immersed in 10% normal goat serum and 0.1% Triton X-100 for 15 min. The primary antisera and dilutions were as follows: mouse anti-pan-cadherin antibody at 1∶200  for cell membrane and mouse anti-Golgin-97 at 1∶200  for Golgi apparatus. After incubation with primary antiserum at 4°C overnight, the cells were rinsed in PBS three times before adding Alexa Fluor 488 and/or Alexa Fluor 594 conjugated secondary antibodies . Endoplasmic Reticulum (ER) was stained with ER-Tracker® Blue-white DPX Probes at 1∶670 dilution  for 10 min at room temperature. The nuclei of cells were counterstained with 4′-6-Diamidino-2-phenylindole  or propidium iodide  for 5 min and rinsed with PBS. Mounted slides were visualized and photographed using a fluorescence microscope . All immunofluorescence cell experiments were performed more than five times and observed more than 200 pairs coupling cells per times with similar results in the study.http://swissmodel.expasy.org/workspace/Although a detailed model of the crystalline structure of human CX30 is still unavailable, its closest homologous protein family member, human CX26 gene (amino acid identity 77%), which has been elucidated by crystallography (PDB entry 2ZW3) to a resolution of 3.5 Å, provides a suitable template http://workbench.sdsc.edu/, San Diego Supercomputer Center) was used to compare amino acid sequences of Cx30 among the b “Insert>Symbol” group in the human CX family. The comparison revealed that p.A40 residue of CX30 protein was highly conserved among the human b “Insert>Symbol” group of the examined CX family members (http://conseq.tau.ac.il/) was also used to study amino acid sequences of CX30 in the CX gene family of all species. After the protein sequence of CX30 was deposited, the system automatically detected homologous sequences of CX30 and conducted multiple alignments. Of the 114 PSI-BLAST hits obtained by the system, 96 were unique sequences. In the next step, the system automatically calculated the 50 sequences with the lowest E-values. The calculation results revealed that p.A40 is highly conserved (Conseq score = 9) and buried in the CX protein  database showed that the CX30 protein subunit, like other CX proteins, contains a short cytoplasmic amino-terminal domain , four transmembrane domains TM; TM1 (amino acid 23∼45), TM2 (amino acid 76∼98), TM3 (amino acid 132∼154), and TM4 (amino acid 155∼192), which is separated by one cytoplasmic loop domain  and two extracellular loops , and a carboxyl-terminal cytoplasmic domain . According to above predictive results, we extrapolated that the p.A40V substitution localized on the putative TM1 domain near the outer membrane .To clari members . A basic protein . Therefo2 labeling reagent and CX30 antibody  or mutant CX30 (CX30A40V-GFP). In cells transiently expressing CX30WT-EGFP, gap junction plaque formation was indicated by WT CX30 expression localized to the cell membranes at points of contact between adjacent GFP-expressing cells. This membrane localization was confirmed by colocalization with pan-Cadherin . SimilarOur previous study found that p.A40V of CX30 is a heterozygous mutation in patients with hearing loss Reports of CX30 and CX26 co-expression in the inner ear To further study the protein-level mechanisms of the p.A40V mutation, a 3D model was constructed for a bioinformatic structural analysis. The analytical results found that the highly conserved residues in this region, including Ala39, Ala40, Val43 and Ile74, comprise a hydrophobic core around Trp44, which stabilizes the CX30 intra-molecular antiparallel organization of the helical structure between transmembrane domain 1 (TM1) and TM2 . At codovia gap junctions has been confirmed by findings that certain CX gene mutations, particularly those of GJB2 and GJB6, cause hearing loss GJB6 is the second most common cause of genetic deafness. A 309-kb deletion has also been reported in the GJB6 gene of hearing loss patients GJB6 (CX30) is known to be involved nonsyndromic deafness by dominant inhibition of the activity of wild-type GJB6 in normal channels, underlining the critical function of GJB6 in normal hearing GJB6, in the patients with nonsyndromic hearing loss GJB6 gene may affect any one or more of these processes. In this article, we have been demonstrated that the missense mutation is belong to group 3 mutation that are functionally deficient owing to trafficking abnormalities.For auditory function, the important role of intercellular communication GJB1 mutant have positions similar to those of the GJB6 p.A40V mutant located at the first transmembrane region and are retained in the ER  or in the Golgi (p.A40V) in HeLa cells GJB2 whose positions coincide with p.A40V of GJB2 have also been detected in the keratitis ichthyosis deafness (KID) syndrome. This study found that the p.A40 residue of CX30 is located at the first transmembrane domain (TM1) and is identical among the human β group of the CX protein family and throughout the evolution of all species. The M1 domain may be a pore-forming domain Gjb2) and Cx30 (Gjb6) are the two most abundantly expressed Cxs in the mouse cochlea Mouse studies suggest that Cx26  syndrome. The mutants have been reported to form hemichannels that open aberrantly, leading to “leaky” cell membranes and show significantly impaired regulation by extracellular CaFigure S1Schematic representation of the domain structure of the CX30 protein with indication of known variants. The black star and arrows indicate the c.119C>T (p.A40V) variant in CX30. M1-4: transmembrane domains; E1-2: extracellular domains; CL: cytoplasmic linking domain; N: N-terminal domain; C: C-terminal domain. [This figure was modified from (TIF)Click here for additional data file.Figure S2ConSeq predictions demonstrated on human CX30 [SWISS-PROT: O95452 (CXB6_Human)], using 50 homologues obtained from the Pfam database (family code: PF00029). The sequence of the CX30 protein is displayed with the evolutionary rates at each site colour-coded onto it (see legend). The residues of the Cx26 sequence are numbered starting from 1. The first row below the sequence lists the predicted burial status of the site (i.e. “b”—buried versus “e”—exposed). The second row indicates residues predicted to be structurally and functionally important: “s” and “f”, respectively. Vertical arrows indicate amino acid codons (p.A40).(TIF)Click here for additional data file.Figure S32 Labeling Kit (A) and anti-CX30 antibody .Expression analysis of CX30WT and CX30A40V in transiently transfected HeLa cells using the FlAsH™-EDT  Fluorescence microscopy of CX30WT HeLa cells shows expression of the wild-type protein in the plasma membranes. (C) In contrast, CX30A40V transfected HeLa cells show expression of the mutated protein near the nucleus. Arrows indicate expression of CX30 protein. Scale bars: 10 µm.(TIF)Click here for additional data file.


During 2009, pandemic influenza A(H1N1)pdm09 virus affected humans on Réunion Island. Since then, the virus has sustained circulation among local swine herds, raising concerns about the potential for genetic evolution of the virus and possible retransmission back to humans of variants with increased virulence. Continuous surveillance of A(H1N1)pdm09 infection in pigs is recommended. Influenza A(H1N1)pdm09 virus, which caused the last influenza pandemic among humans, is a unique reassortant derived from swine influenza viruses of the triple reassortant swine North American lineage and the avian-like swine Eurasian lineage , and titers were determined by using hemagglutination-inhibition (HI) assays (>20); the range of positive titers was 40–640, and 54.2% of the samples expressed high HI titers (160–640). Of the 98 serum samples, 5 reacted at low titer and with only 1 European A (H1N1) swine virus (titer <20), i.e., >4 dilutions lower than for A(H1N1)pdm09 virus, indicating cross reactivity (During a first step (November 2009–February 2010), seroprevalence rates for A(H1N1)pdm09 virus were assessed in 120 breeding pigs (>4 years old) from 57 farms. Blood was obtained from randomly selected pigs at the only slaughterhouse on the island, where pigs are held for ) assays . Ninety-activity .In a second step pdm09 infection was no longer detected among humans), we tested whether the virus was still circulating among pigs born that year. To obtain nasal swab and blood samples for testing, we randomly selected 390 fattening pigs (25–27 weeks old) at the slaughterhouse; the pigs originated from 45 farms. At the time of sampling, the veterinary surveillance system did not report any clinical signs suggesting virus circulation among herds. However, ≈3.5% of the serum samples (9% of tested farms) contained antibodies to A(H1N1)pdm09 virus (HI titers 20–160). Nasal swab specimens from 6.7% (26/390) of pigs were positive for A(H1N1)pdm09 virus as determined by using a specific real-time reverse transcription PCR (rRT-PCR); the pigs originated from 13 (28.8%) farms pdm09 virus infection was established on 3 farms (farms A–C). In June 2010, fattening pigs on farm A were seronegative for A(H1N1)pdm09 virus. In July, when acute respiratory disease was reported among pigs, 12 of 39 fattening pigs (18–21 weeks old) sampled on farm A were still seronegative for A(H1N1)pdm09 virus; however, rRT-PCR results were positive for A(H1N1)pdm09 virus. Four weeks later, when pigs had recovered from influenza, only 7.7% (3/39) of sampled pigs on farm A had rRT-PCR results positive for A(H1N1)pdm09 virus, and all 39 were seropositive for the virus. High rates of rRT-PCR positivity were also noted for pigs on farms B (17/30 pigs) and C (6/15 pigs). Two A(H1N1)pdm09 strains (A/Sw/LaReunion/0167/10 and A/Sw/La Reunion/110194/10) were isolated from pigs on farms A and B, respectively.Four influenza virus strains were isolated from pigs, and all induced a cytopathic effect and displayed hemagglutinating activity on chicken erythrocytes; all 4 were confirmed as A(H1N1)pdm09 virus by specific rRT-PCRs. In addition cross-HI assays . Genome In a third step , 3 other sampling campaigns were conducted at the slaughterhouse, including 831 fattening pigs from 104 farms. Nasal swab samples for 7 (8.4%) pigs from 3 (2.9%) farms still had rRT-PCR–positive results. However, serologic analyses revealed that pigs on ≈40% of the farms (distributed throughout the island) were seropositive for A(H1N1)pdm09 virus, indicating continuing circulation of the virus in swine herds .Consistent with findings elsewhere pdm09 virus. First, the reassortant pandemic virus contains genomic segments originating from swine influenza viruses established in pigs (Mycoplasma hyopneumoniae and Pasteurella multocida were co-detected on farms with pigs with signs of infection (data not shown). Co-infection with swine influenza virus and these bacteria is known to contribute to severe respiratory disorders among pigs; thus, these bacteria may have enhanced pathogenicity of A(H1N1)pdm09 virus on affected farms pdm09 virus will decline over time when the virus is no longer circulating among humans, persistence of the virus in an animal reservoir raises concerns about the risk for genetic evolution of the virus and retransmission back to humans of variants with potentially increased virulence. As an example, during the 2011 austral winter, only influenza A(H3N2) and B viruses were recorded (


Uncoupling Protein 2 (UCP2) dan Uncoupling Protein 3 (UCP3) is a mitochondrial transmembrane carriers which uncouple the transport of protons across the inner mitochondrial membrane from electron transport and the synthesis of ATP from ADP. The UCPs play a role in energy homeostasis and have considered to be candidate gene for controlling obesity and insulin resistance. The aim of our study is to analyze UCP2 and UCP3 polymorphisms as risk factors of insulin resistance in obese adoslescent.A case control study was conducted in Junior High School in Semarang, Central Java, Indonesia during 2007- 2008. Seventy five subjects included in this study. Height, weight and body mass index (BMI) were measured. Insulin resistance was estimated by using the homeostasis model assessment (HOMA-IR) score which calculated as fasting insulin (microU/mL) x fasting glucose (mmol/L)/22.5. Three polymorphic sites i.e. UCP3 -55c/t, UCP3 Y210Y, and UCP2 A55V were investigated by using the Restriction Length Fragment Polymorphism. The data were analyzed by 2x2 table and odd rasio with 95% confidence interval.There were 38 obese  and 37 normoweight  subject with the mean age was 13.2 (SD 0.32) years. Of 38 obese, there were 3 (7.9%) subject had insulin resistance. None of normoweight subjects had insulin resistance. There was a significant correlation between BMI and insulin resistance . Of 38 obese subject, C allele of UCP2 A55V and C allele of UCP3 Y210Y are a risk factors of insulin resistance , whereas UCP3 -55c/t was not considered as risk factors of insulin resistance.Our study suggest that C allele of UCP2 A55V and C allele in UCP3 Y210Y are a risk factors of insulin resistance in Indonesian obese adolescent. Larger studies is needed to prove the role of UCPs in controlling insulin resistance.


Background: Sar1 mediates the onward transport of ER cargo.Results: Sar1B promotes VLDL secretion, whereas Sar1A antagonizes this activity, and a deficit of both reduces cholesterol biosynthesis.Conclusion: Sar1B independently of and through its lipoprotein secretion function promotes the expression of genes regulating cholesterol biosynthesis.Significance: Sar1B-mediated transport activities contribute to both the functional integrity of the ER membrane and blood cholesterol levels. de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions.Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and  It m–46–i.e. Sar1:H79G) have defined several aspects of the COPII vesicle assembly process  using gene-specific primers. FLAG epitope tags (Asp-Tyr-Lys-Asp4-Lys) were fused in-frame to the carboxyl termini of cDNAs and juxtaposed to a terminator codon. PCR and appropriate restriction sites were used to manipulate the SAR1 sequences. Site-directed mutagenesis was performed by a standard two-step PCR-based strategy .Knockdowns were performed with pre-designed siRNA (Qiagen) using HiPerFect transfection reagent (Qiagen) according to the manufacturer's fast forward protocol. Volumes of transfection reagents and concentrations of siRNA were optimized for each pre-designed FlexiTube siRNA (Qiagen). The best results were obtained using a final concentration of 5 n2 in DMEM containing 20% heat-inactivated FCS , 4500 mg/liter d-glucose, 2 mml-glutamine (Invitrogen), and 1% penicillin/streptomycin (Invitrogen). CHO cell lines were cultured in Ham's F-12 media (Sigma) containing 10% heat-inactivated FCS, 2 mml-glutamine, and 1% penicillin/streptomycin. Blasticidin  and Zeocin  were added to the media of cells stably transfected with the tetracycline repressor (pcDNA6/TR) and operon (pcDNA4/TO) vectors (Invitrogen).McArdle-RH7777 cells were cultured at 37 °C, 5% COl-[35S]methionine (ICN Flow) at 37 °C, cells were incubated with complete DMEM containing 1 μg/ml tetracycline between 24 and 48 h and then in serum-free media supplemented with 0.8 mm oleic acid (Sigma) complexed to 3% fatty acid-free BSA overnight and cysteine/methionine-free media (Invitrogen) containing oleate for 30 min. After removal of label, incubations were continued in serum-free media supplemented with 0.8 mm oleic acid complexed to 3% fatty acid-free BSA for 2 h. Conditioned media were cleared by low speed centrifugation and concentrated to 500 μl in Amicon Ultra-4 centrifugal filter units with Ultracel-30 membranes (Millipore). ApoB was immunoprecipitated by standard methodology . After removal of label, incubations were continued in serum-free medium for 2 h. Cell media were harvested, mixed with protease inhibitors , and cleared by low speed centrifugation. Proteins were precipitated by incubation with TCA for 15 min on ice. Precipitates were washed in ice-cold acetone and resuspended in I.P. buffer containing protease inhibitors .For total protein secretion measurements, cells were incubated in methionine-free medium for 45 min at 37 °C and labeled for 1 h with 125 Ci/ml 35S-labeled apoB lipoproteins were determined by gradient ultracentrifugation. The sucrose gradients were formed as described  and a mixture of protease inhibitors (Roche Applied Science). Proteins were size-fractionated on either 10 or 4–12% gradient NuPage BisTris precast gels (Invitrogen) and transferred to nitrocellulose membranes (Whatman). Membranes were blocked in 5% (w/v) nonfat milk powder before probing with the specified antibodies. The antibodies used were as follows: rabbit polyclonal anti-Hmgcs1 antibody (Proteintech); anti-Lss antibody (Proteintech); anti-Dhcr7 antibody (Abcam); anti-ACSL5 (Proteintech); anti-Sec13 antibody (Abcam); anti-Sec31a antibody, anti-Sar1a antibody, anti-Sar1b antibody, and anti-Srebf2 antibody (Proteintech); anti-ATF6 (Abcam); and mouse monoclonal β-actin or β-tubulin antibodies (Sigma and Abcam). Immunoreactive products were visualized and quantified after labeling with appropriate fluorescent dye-labeled secondary antibodies (LI-COR Biosciences) using the Odyssey imaging system (LI-COR). Apparent molecular masses were estimated using the Novex Sharp Protein Standard (Invitrogen) and the band sizing application in Odyssey software (LI-COR Biosciences).supplemental Table S1). Total RNA was isolated from cell lysates using the RNeasyPlus kit (Qiagen). Cells were homogenized using QIAshredder columns (Qiagen). RNA samples were quantified by a NanoDrop 1000 spectrophotometer (Thermo Scientific). RT-qPCRs were performed in triplicate (with a no RT control) with SYBR Green kits from Qiagen or Stratagene, as recommended by the manufacturers. Data were analyzed with either SDS 2.3 software (Applied Biosystems) or MxPro (Stratagene). Gene expression levels, normalized to Ppia, were calculated by the relative 2−ΔΔCt method (Pre-designed gene primers were purchased from Qiagen (Quantitect primer assay), unless stated otherwise (t method .i.e. treatment-naive). To minimize variability in sample processing and handling, biopsy samples were collected immediately and snap-frozen in liquid nitrogen. All subsequent sample processing was performed by the same individual using the same standardized protocol. Tissue was homogenized in MagNA Pure LC RNA Isolation Tissue Lysis Buffer (Roche Applied Science), using a MagNALyser instrument (Roche Applied Science), and using parameters that were optimized on comparable samples prior to commencing the study. Total RNA was prepared using TRIzol according to the manufacturer's manual (Invitrogen), quantified by a NanoDrop 1000 spectrophotometer (Thermo Scientific), and reverse-transcribed into cDNA using a cDNA synthesis kit from Bio-Rad. RT-qPCR was performed in triplicate with 50 ng of cDNA, 200 nm of each primer, and 7.5 μl of SYBR Green mix (CE Biotech). PCRs were performed on a CFX system (Bio-Rad). Gene expression levels normalized to 36B4 were calculated by the relative 2−ΔΔCt method  biopsies were taken near the Treitz' ligament in 17 obese male subjects with no history of disease or any medication use /(n − 2), where R is the correlation coefficient, and n denotes the number of samples.RT-qPCR on human liver RNA  was performed with a SYBR Green kit (Qiagen). Correlations between mRNA levels in human liver samples , 63 weree.g. flat baseline between 18 S and 28 S ribosomal RNA peaks; RIN score >7) using the RNA 6000 Ladder (Ambion), RNA 6000 NanoChip (Agilent), plus an Agilent 2100 bioanalyzer . cDNA was synthesized from total RNA using the one-cycle eukaryotic target labeling assay kit (Invitrogen and Affymetrix). Double-stranded cDNA was purified with the GeneChip® sample cleanup module. Biotin-labeled cRNA was prepared using the BioArray High Yield RNA transcript labeling kit (Enzo) and purified with the in vitro transcription cRNA GeneChip® sample cleanup module. RNA quality was verified with the Agilent Bioanalyzer 2100 system and by hybridization to test arrays (Affymetrix). Biotinylated cRNA (20 μg) was hybridized to GeneChip rat genome 230 2.0 arrays (Affymetrix) containing 31,099 probe sets. All cRNA samples were prepared using the same standard protocol.A minimum of three technical replicates were used for each of two Sar1B:H79G-FLAG cell lines, two control cell lines, two Sar1B-FLAG cell lines, and one Sar1A-FLAG cell line. Cells were cultured in complete DMEM containing blasticidin  and Zeocin . Twenty four hours prior to harvesting, 1 μg/ml tetracycline was added to the culture media. Total RNA was extracted with the RNeasy Plus mini kit (Qiagen). RNA quality was verified  algorithm (GSE52969). The differentially expressed probe sets were examined for enrichment of functionally related Gene Ontology terms using the Database for Annotation, Visualization, and Integrated Discovery  . Raw intensity values were normalized using the Loess normalization, Affymetrix MAS 5 and summarization algorithms. Differences in probe-set values were determined on the normalized data using the lgorithm  at specicrf.gov) , 66.4 cells/cm2 in 9-cm plates and grown for 2 days in DMEM containing 20% FCS, 1% penicillin/streptomycin, and 1 mm glutamine at 37 °C under 5% CO2. Lipid synthesis was measured over the final 5 h of the incubation by the addition of 2 mm [14C]acetate  (0.8 μCi/ml). Cell media were aspirated for analysis of lipid secretion. Cells were washed in PBS and harvested by trypsinization and centrifugation. Cell pellets were washed in PBS and lipids extracted by addition of 4 ml of chloroform/methanol (2:1). Water (1 ml) was added to the extract and mixed by vortexing. The suspension was centrifuged for 5 min, and the lower solvent layer was taken for analysis. The upper aqueous layer was re-extracted with a further 4 ml of chloroform/methanol, and the two extractions were combined. This was dried down under N2 and resuspended in a minimal volume of hexane (30 μl). Cell medium (4 ml) was extracted as above, using 16 ml of chloroform/methanol (2:1). Lipids were separated by thin layer chromatography on Silica Gel 60 plates (VWR) using petroleum ether/diethyl ether/acetic acid (60:40:1) as the mobile phase and visualized by iodine staining. Each run included standards of cholesterol, fatty acids, triglycerides and cholesterol esters. Lipid synthesis was measured by scraping of spots into vials followed by liquid scintillation counting. Plates were also visualized by autoradiography to determine that no major lipid product was missed.Cells were seeded at a density of 1 × 10t test or one-way analysis of variance followed by the Bonferroni post hoc test using PASW Statistics version 18.0 software , where appropriate. Figures were generated using Graphpad Prism 4.All data are expressed as means ± S.E. Statistical analyses were performed using unpaired Student's i.e. Sar1A:H79G and Sar1B:H79G) isoforms that cannot hydrolyze GTP to support completion of COPII vesicle assembly (Sar1a mRNA is the predominant species expressed in these cells (11.6-fold higher than Sar1b). The Sar1A cell lines contained 5.26 ± 0.48- and 2.41 ± 0.48-fold, respectively, more recombinant Sar1 than the Sar1B and Sar1B:H79G cell lines.To establish whether Sar1B promotes only the secretion of apoB48-containing lipoproteins, we stably transfected McArdle-RH7777 cells that can secrete both apoB48 and apoB100 lipoproteins with constructs encoding Sar1A-FLAG, Sar1B-FLAG, or constitutively active Sar1-FLAG (assembly . DespiteA). By contrast, Sar1B overexpression increased their secretion by >3-fold (B). Their densities were not decreased (C and D) with both the apoB48- and apoB100-containing lipoproteins exhibiting their expected densities (E); however, the effect was more pronounced for apoB100 than apoB48 (secretion 3.49 ± 1.5% (apoB100) and 20.65 ± 3.97% (apoB48) of control cells, respectively; p = 0.0016 for difference). Total protein secretion was also decreased in the Sar1B:H79G cell lines (F), but this reduction was significantly less than that observed for apoB48/100 (A). Collectively, these data show that Sar1B promotes secretion of both apoB48 and apoB100 lipoproteins, whereas Sar1A overexpression has the opposite effect, preferentially blocking the secretion of the more lipid-laden particles.Sar1B:H79G overexpression virtually abolished the secretion of both apoB48 and apoB100 lipoproteins A. By con >3-fold B. Their ecreased , C and Densities . Sar1A oensities E; howevell lines F, but thoB48/100 A. CollecApoB and Mttp, we measured the levels of their RNAs by RT-qPCR in our McArdle-RH7777 cell lines plus McArdle-RH7777 cells transfected with Sar1 siRNAs. Individual knockdown of the Sar1 isoforms were isoform-specific, and there were no increases in the mRNA (A) or protein (B) levels of the nontargeted isoform. The amounts of Sar1a protein in cells transfected with Sar1a and Sar1a plus Sar1b siRNAs were 28.16 ± 6.36 and 32.88 ± 8.70%, respectively, of that in cells transfected with the scrambled siRNA control (B). Sar1b knockdown also substantially reduced in Sar1b protein and in the majority of samples to undetectable levels (B). This was due to the Western blot analysis returning weaker signals for Sar1b than Sar1a, consistent with RT-qPCR data (Sar1b mRNA 11.6-fold lower than Sar1a mRNA), plus the efficiency of Sar1b knockdown in reducing Sar1b protein (≥70% in the rare knockdown sample where it could be quantified).To determine whether Sar1 also had isoform-specific effects on the expression of the mRNA A or prot protein B levels  control B. Sar1b e levels B. This wSar1a mRNA (4.22 ± 0.29-fold compared with control cells) and protein (C), it reduced ApoB and Mttp mRNA (D). Levels of the control gene, Mapk1, were not altered (D). In the complementary analyses, native Sar1B overexpression increased ApoB mRNA (D), while having no effect on Mttp (D) and Sar1a  mRNAs; and Sar1b knockdown reduced ApoB and Mttp mRNA (E) and had no significant effect on Sar1a mRNA (A). In the control analysis, ApoB and Mttp knockdown (F) did not reduce Sar1b mRNA (G). Interestingly, we found that Sar1A overexpression decreased Mttp mRNA levels, although Sar1a knockdown did not increase Mttp mRNA (E). However, in the double knockdown experiment, decreased Sar1a did prevent the fall in Mttp (and apoB) associated with Sar1b knockdown (E). Thus, collectively these data indicate that Sar1 has isoform-specific effects on ApoB and Mttp mRNA levels. Moreover, because a higher level of MTTP activity is required for producing apoB100 than apoB48 lipoproteins  and a pooled (three donors) liver sample. By contrast, the predominant SEC23 (A) and SEC31 (A) isoforms expressed in these two tissues displayed more comparable mean fold differences; in the intestinal biopsies, SEC23A and SEC31A were 5.0 ± 0.9- and 43 ± 12-fold higher than their respective B isoforms; and in the pooled liver sample, the corresponding values were 3.2 and 29.4.We found that SAR1B, but not SAR1A, mRNA levels and APOB/MTTP mRNA. Thus, in a large dataset comprising 427 individual liver samples, the correlation coefficient (R) between SAR1B and APOB mRNA was 0.301 (p = 8.3 × 10−9), and in the second dataset, comprising 75 samples, this result was replicated . Likewise, R values between SAR1B and MTTP mRNA were 0.268 (p = 2.5 × 10−8) in dataset 1 and 0.346 (p = 2.4 × 10−3) in dataset 2. SAR1A displayed no positive correlation with either APOB or MTTP mRNA levels; R values for the SAR1A/APOB correlations were 0.003  and −0.012  and between SAR1A and MTTP, −0.179 (p = 2.3 × 10−4) and −0.019 (p = NS). Notably, the weak inverse correlation between SAR1A and MTTP mRNA levels in dataset 1 is consistent with the effect of Sar1A overexpression on Mttp mRNA levels in McArdle-RH7777 cells (D), plus the Mttp mRNA findings in the set of Sar1 knockdown experiments (E).We also found positive correlations between hepatic 77 cells D, plus teriments E.e.g.D and E) and the previous characterization of the COPII vesicle assembly process . In addition, we confirmed that the respective 2.73 ± 0.15- and 2.90 ± 0.47-fold increases in Sec13 and Sec31a mRNA associated with Sar1B:H79G overexpression  were congruent with estimated rises in Sec13 (1.7 ± 0.20-fold) and Sec31a (4.72 ± 0.94-fold) protein.In view of the lipid transport functions of apoB, we determined whether constitutively active Sar1B altered the expression of genes regulating triglyceride, fatty acid, and cholesterol homeostasis. To this end, we performed a microarray RNA analysis on the McArdle-RH7777 cell lines and validated the robustness of the results by measuring A). In the control analyses (i.e. native Sar1 overexpression), fewer probe sets were uniquely differentially expressed as follows: three in the Sar1A- and 70 in the Sar1B-overexpressing cell lines . The Sar1B:H79G probe set contained an over-representation of genes assigned the Gene Ontology terms “Cholesterol and Lipid Biosynthetic and Metabolic Processes” . We therefore collated the expression values for genes active in fatty acid, triglyceride, and cholesterol metabolism, and verified the mRNA levels of representative genes in an independent experiment by RT-qPCR .We identified 701 probe sets that had altered expression values (FDR <0.000215) in the Sar1B:H79G cell lines only A. In theocesses” , althougA and E), and increased apoB-secretion from Sar1B cells (B), we found that the levels of most transcripts encoding proteins active in fatty acid and triglyceride metabolism were unaltered by increases in Sar1A expression in the Sar1A and Sar1B:H79G (C) cell lines and by Sar1B overexpression . By contrast, constitutively active Sar1B decreased the levels of most mRNAs encoding enzymes on the cholesterol biosynthetic pathway , whereas overexpression of native Sar1A and -B did not .Despite reduced secretion of apoB lipoproteins from the Sar1B:H79G and Sar1A cell lines , A and E1B cells B, we four1B:H79G C cell liC, 1st and 2nd panels), whereas Dhcr7, which converts 7-dehydrocholesterol to cholesterol and 7-dehydrodesmostrel to 24-dehydrocholesterol , was increased >2-fold . In a control analysis, Sar1B:H79G did not alter RNA  or protein levels of Acsl5 , a key regulator of fatty acid metabolism (E), did not reduce Hmgcs1 and Lss protein  or increase Dhcr7 protein . Rather Hmgcs1 protein was increased .Western blot analysis of Sar1B:H79G cell lysates revealed markedly reduced levels of Hmgcs1 and Lss protein . However, no increase in Dhcr7 protein was observed, and in marked contrast to Sar1A and Sar1B:H79G overexpression, Ascl5 protein was reduced (D). Thus, these data indicate that overexpression of Sar1A, Sar1B, and Sar1B:H79G have distinct effects on Hmgcs1, Dhcr7, and Acsl5 protein levels and that only Sar1B:H79G significantly reduced levels of mRNA encoding cholesterol-biosynthetic enzymes .Sar1B overexpression had a variable impact on Hmgcs1 protein levels and only significantly reduced Lss D. Howeve reduced D. Thus, ApoB and Mttp did not significantly reduce mRNA levels of cholesterol pathway-encoding enzymes . Thus, these data substantiate the evidence from the Sar1A gene expression  and apoB secretion results (E) that partial ablation of cholesterol/cholesteryl ester secretion via the apoB-containing lipoprotein axis .In a further control experiment, we found that knockdown of  results E that paein axis , 72, 73 Sar1A mRNA levels were respectively increased by 18.68 ± 2.04- and 22.13 ± 1.12-fold in the Sar1A and Sar1A:H79G cell lines; and the corresponding Sar1B values for the Sar1B and Sar1B:H79G cell lines were 21.17 ± 2.87 and 38.08 ± 3.71. Sec and control gene expression was much more modestly affected (A). In these non-apoB lipoprotein-producing cell lines, the overexpression of native Sar1A and Sar1B reduced cholesterol biosynthesis gene expression, but to a lesser extent than the control genes (A and B). By contrast, constitutively active Sar1A or Sar1B overexpression markedly reduced mRNA levels of the cholesterol gene set (B). Hence, these data support the proposition that the reduced levels of most transcripts encoding enzymes for cholesterol biosynthesis in the McArdle-RH7777 cell lines expressing Sar1B:H79G are partially separable from the apoB lipoprotein secretion defect.CHO cells do not have the machinery to produce apoB-containing lipoproteins . We theraffected A. In theol genes , A and Bgene set B. Hence,B) also suggested that both Sar1 isoforms modulate the levels of mRNAs encoding cholesterol biosynthetic enzymes. We examined this further in McArdle-RH7777 cells by determining the effects of Sar1a and Sar1b knockdown on mRNA levels of eight representative members of this gene set. All were decreased (C). Sar1b knockdown generally produced smaller reductions than knockdown of Sar1a, the predominant species in McArdle-RH7777 cells. In the double knockdown, Hmgcs1 and Dhcr7 mRNA levels were significantly lower than in the individual knockdowns (C). However, Hmgcs1 protein levels were not significantly decreased by either individual or combined knockdown of Sar1a and Sar1b (D). In comparison, Lss protein was significantly reduced by both individual and combined Sar1a and Sar1b knockdown (E). Combined  Sar1a and Sar1b knockdown also produced a nonsignificant rise in Dhcr7 . Thus, collectively, these data indicate that both Sar1A and Sar1B modulate the levels of mRNAs encoding cholesterol biosynthetic enzymes, supporting the CHO cell findings . Substantiating this conclusion, we also found that in human liver samples, SAR1A and SAR1B mRNA levels exhibited no isoform-specific correlations with 20 of the 21 mRNA species encoding cholesterol biosynthetic enzymes . The only isoform-specific correlation was between that of SAR1B and SC5DL (sterol-C5-desaturase), and both of these genes had expression values that exhibited positive correlation with APOB and MTTP mRNA levels .The gene expression data from CHO cell line experiments B also suecreased C. Sar1b ockdowns C. Howevend Sar1b D. In comnockdown E. CombinB and 4C) and associated protein changes  translated into reduced de novo cholesterol synthesis. As estimated from the incorporation of [14C]acetate into cholesterol, Sar1B:H79G overexpression markedly reduced the rate of de novo cholesterol synthesis (A). Additionally, [14C]cholesterol secretion was reduced (B) but to a lesser extent than de novo cholesterol synthesis. The estimated rate of incorporation of [14C]acetate into nonesterified fatty acids (NEFA) was also reduced (C), but this was to a much lesser degree than de novo cholesterol synthesis, and there was no corresponding reduction in 14C-labeled NEFA secretion (D). Consistent with comparable transcription profiles , the Sar1A and Sar1B cell lines displayed similar rates of de novo cholesterol synthesis (A), both of which were significantly higher than that of the Sar1B:H79G cell lines.Next, we wanted to establish whether the reductions in “cholesterol” gene expression B and 4C ynthesis A. Additi reduced B but to  reduced C, but thecretion D. Consisynthesis A, both oSar1a and Sar1b did not reduce the estimated rates of de novo cholesterol synthesis (E). By contrast, combined Sar1a and Sar1b knockdown had a significant impact reducing cellular [14C]cholesterol by 25.33 ± 8.04% (E), and this was paired with a small, albeit nonsignificant, reduction in the estimated rate of [14C]cholesterol secretion (F). The estimated rate of incorporation of [14C]acetate into NEFA was also reduced by combined Sar1a and Sar1b knockdown but by no more than individual Sar1a and Sar1b knockdowns (G and H). Thus, these data, combined with the results in B–D, and B–F, and previous findings  and a significant increase in the level of the 68-kDa processed polypeptide (B and C). Levels of the control protein ATF6 were not altered . Thus, these results indicate that there was sufficient endogenous Sar1 in the Sar1B:H79G cell lines (C) to support the ER export of both Srebp2 and ATF6 precursors.We found that Sar1B:H79G overexpression led to a small, but nonsignificant, rise in the level of precursor Srebp2 A and a sypeptide , B and Cll lines C to suppD–F) or Scap and Srebp2 mRNA expression . Likewise, combined Sar1a and Sar1b knockdown did not decrease Srebp2 protein levels or its processing (D–F). By contrast, knockdown of Sar1b (minority Sar1 species in McArdle-RH7777 cells), which had no impact on de novo cholesterol synthesis (E), significantly reduced  the level of processed Srebp2 (G–I) and Scap mRNA . In the control analysis, Sar1b knockdown did not reduce the amount of the non-sterol-responsive transcription factor ATF6 .Sar1a knockdown did not reduce either Srebp2 protein levels , D–F or ocessing , D–F. Byynthesis E, signifd Srebp2 , G–I andde novo cholesterol synthesis and their reliance on the common and individual activities of the two Sar1 isoforms. We provide strong evidence that Sar1B promotes the secretion of triglyceride-rich, apoB-containing lipoproteins from the liver, which would neatly explain the counter-intuitive observation that some CMRD children develop hepatic steatosis, despite severe intestinal fat malabsorption  that features in CMRD , bind considerably more lipid than apoB48  have been found within the abluminal space of CMRD patients' enterocytes , implyinn apoB48 , 93, 94.ApoB expression (D) but not of genes regulating triglyceride synthesis  and that Sar1B promoted the production of increased numbers of apoB lipoproteins rather than of more lipid-laden ones (C and D). In comparison, Sar1B mutations in CMRD culminate in the production of abnormally large chylomicrons , well bSAR1A, SAR1B mRNA levels are much higher in the upper gastrointestinal mucosa than in liver. Nonetheless, it is apparent that SAR1B contributes in a major way to hepatic lipid metabolism given the combination of our apoB lipoprotein studies , the finding of hepatic steatosis in some children with CMRD  paired with the expected corresponding reduction in levels of mRNAs encoding enzymes active in cholesterol biosynthesis (C).We found that relative to ith CMRD , 17, 91,ocessing H paired ynthesis C.supplemental Table S5). Moreover, even though constitutively active Sar1B blocked apoB lipoprotein secretion (A), it did not reduce the expression of most members of these two gene sets, suggesting that the intracellular transport of Srebp1, the best characterized transcription factor regulating fatty acid and triglyceride gene expression (96–C), we very likely created the means for allowing the onward transport of some ER cargo, while facilitating detection of Sar1B-specific phenotypes.We found that Sar1 had no isoform-specific effects on the expression of genes regulating triglyceride or fatty acid metabolism , they did reduce de novo cholesterol biosynthesis, albeit to a much lesser extent than Sar1B:H79G overexpression. It seems particularly pertinent that Sar1A overexpression, which markedly decreased apoB lipoprotein secretion (E), was associated with increased Hmgcs1 protein levels, and Sar1B overexpression was associated with reduced Lss protein. Conceivably, this counter-intuitive rise in Hmgcs1 protein (but not mRNA) could reflect a response to the diversion of acetyl-CoA (B) away from the cholesterol biosynthesis pathway toward an alternative metabolic pathway(s), for example, pathways promoting the acetylation of ER resident and ER-transiting proteins  and that this reduction was paired with increased, rather than decreased, Dhcr7 protein levels is of interest . Dhcr7 deficiency in embryonic mouse brains leads to a compensatory rise in Hmgcs1 protein . Likewise, the expected reciprocal relationship between reduced de novo cholesterol synthesis and increased Srepb2 processing (B and C) was sustained. However, this increased processing was insufficient to secure the expected corresponding increases in cholesterol biosynthetic gene expression, as judged by mRNA measurements.The observation that Sar1B:H79G overexpression reduced the levels of most transcripts encoding cholesterol biosynthetic enzymes .There are several possible explanations that could account for the effect of the Sar1B:H79G mutation on the levels of mRNA encoding cholesterol metabolism genes. For instance, it could reflect reduced Srebp2-mediated activation of these genes, for example, as a result of dimerization of Srebp2 with Srebp1c and/or another transcriptional co-factor , 104. NeC), but despite this, estimated rates of de novo cholesterol synthesis and secretion were not reduced. Consistent with this, Srebp2 processing was not increased. Thus, our results indicate that in our cell system there was sufficient Sar1  to initiate the assembly of pre-budding COPII complexes for ER export of Srebp2 and that factors other than reduced Srebp2 processing underlie the reduced mRNA levels of the cholesterol gene set. By contrast, the reduced processing of Srebp2 associated with knockdown of Sar1b (minority species) may explain some of the reduction in the levels of mRNA encoding cholesterol biosynthetic enzymes. Hence, the observed cholesterol/Srebp2/mRNA differences between the Sar1B:H79G overexpressing cells and cells deficient for Sar1a or Sar1b indicate that the processes regulating de novo cholesterol synthesis in apoB lipoprotein-producing cells may be somewhat more complex than in other cell types. Furthermore, modulation of Sar1A and Sar1B levels in such cells affects processes regulating the accessibility of de novo synthesized cholesterol within the ER membrane  protein. It is also very striking that the severe hypocholesterolemia of CMRD differs from abetalipoproteinemia , increased de novo cholesterol synthesis and the production of cholesterol-enriched VLDL, culminating in markedly raised plasma levels of total and LDL cholesterol (B) occur on the ER membrane  onto assembling apoB lipoproteins, thereby removing product inhibition (With respect to understanding the role of hepatic l levels . Conversl levels B, increalesterol . Becauselesterol B occur omembrane –28, it ii et al.  have shoi et al. , and Tem (et al.  demonstrhibition . Thus, ihibition , whereashibition , 81, 90.hibition .


Hyperzincaemia and hypercalprotectinaemia, a rare condition within the spectrum of autoinflammatory diseases, is associated with recurrent infections, hepatosplenomegaly, arthritis, anemia, cutaneous inflammation, and failure to thrive. So far, no genetic cause has been identified in these patients. While the clinical appearance is heterogeneous, all affected individuals present with extremely elevated S100A8/S100A9  serum concentrations ).PSTPIP1 and MEFV was performed in 7 hyperzincaemia and hypercalprotectinaemia patients to identify disease-causing mutations.The clinical phenotype of 8 patients was characterized. Screening of candidate genes Serum concentrations of S100A8/S100A9 were analyzed by an ELISA assay in 8 patients with hyperzincaemia and hypercalprotectinaemia and compared to PAPA patients with and without treatment. Candidate exons were amplified by PCR and sequenced on an ABI 3130 Genetic Analyzer.PSTPIP1 gene. S100A8/S100A9 concentrations were extremely elevated in these patients (0.9-12 g/l) compared to seven patients presenting with classical PAPA symptoms (0.02-0.35 g/l), whose levels again were significantly higher compared to normal controls.Seven of the eight patients were heterozygous carriers of a glutamic acid 250 (GAG)→lysine (AAG)/p.Glu250Lys/E250K substitution in exon 11 of the PSTPIP1 gene.The PSTPIP1 E250K mutation causes an autoinflammatory disorder known as hyperzincaemia and hypercalprotectinaemia. The disease causes a heterogeneous spectrum of symptoms that only partially overlaps with the presentation of the classical PAPA syndrome. Elevated S100A8/A9 levels are a common hallmark and biomarker of disorders caused by mutations in the


Aberrant inflammasome priming and dysregulated secretory pathways contribute to parallel IL-18 and S100A12 hypersecretion from neutrophils in instable FMF.The study was performed to assess the ex vivo inflammasome activity in granulocytes from patients with unstable FMF. Furthermore, the secretion of S100A12 molecules after various kinds of cell stimulation was assessed.6 turkish patients with the clinical diagnosis of FMF exhibiting homocygous or combined heterozygous mutations within the MEFV gene were included. Patients still exhibited clinical symptoms and elevated inflammation markers despite sufficient colchicine therapy (“instable disease”). Healthy probands served as controls. Their health status was assessed by a standardized questionnaire. All patients and controls gave written consent.25 – 30 ml blood was drawn and PBMC and granulocytes separated by a two density gradient centrifugation. 5 x 106 cells were stimulated with (i) mock, (ii) PMA (10nM), (iii) LPS (10ng/ml) and (iv) LPS (10ng/ml) + ATP (1mM) (later substance for the last 30 minutes). In a similar approach cells were treated with additional colchicine (5μg/ml) for the whole incubation time. Supernatant was gained and frozen at -20oC after 5 hours. ELISA for S100A12, IL-18 and caspase-1 were performed according to standard protocols.At the time of blood drawing high sensitivity CRP was measured.Compared to controls even unstimulated granuloytes from FMF patients with instable disease secreted significantly more S100A12 , IL-18  and caspase-1 . Stimulation also induced enhanced secretion of S100A12 , IL-18  and caspase-1 . Furthermore, supplementary colchicine significantly suppressed the hypersecretion of S100A12, IL-18 and caspase-1.The spontaneous release of IL-18 and caspase-1 demonstrated the constant inflammasome activity in patients with instable FMF. It was not be further induced by LPS/ATP stimulation. S100A12, although not processed by the inflammasome, was also secreted in high amount from FMF neutrophils, irrespective of further stimulation. Taken together, these data indicate that FMF neutrophils show a spontaneous hypersecretion of S100A12 and IL-18 which is only partially related to aberrant inflammasome activation  but may also dependent on dysregulated alternative secretion (inhibited by colchicine in vitro).None declared