Moreover, CLint values of CYP3A4-POR  were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR  on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication.Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR  were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ∼70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CL Members of cytochrome P450 monooxygenase superfamily (CYP) are known to catalyze the biotransformation of xenobiotics, including therapeutic drugs, carcinogens and many other environmental chemical compounds VKORC1_1639A>G, CYP2C9*2, CYP2C9*3, CYP4F2 rs2108622, and chronic aspirin therapy), three common POR SNPs  were also significantly associated with variations in warfarin maintenance dose Apparent variation of CYPs-mediated drug metabolism could alter therapeutic efficiency and toxicity of many drugs in clinical treatments, which was thought to be caused by CYPs polymorphisms in early studies POR is a flavoprotein with 680 amino acids (NCBI NP_000932.3) localized in the endoplasmic reticulum (ER) as the sole electron donor for all microsomal CYP enzymes, hemeoxygenase and fatty acid elongase NADPH-cytochrome P450 oxidoreductase (POR) catalyzes the transfer of electrons from nicotinamide adenine dinucleotide phosphate (NADPH), via two flavin cofactors namely, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), to various cytochrome P450s and catalyzes the one-electron reduction of many drugs and xenobiotics in vitro (reconstituted) systems, using N-terminal end-deleted CYPOR by bacterial system (prokaryocytes) since 2008.In fact, the majority of studies on POR variants were initially focused on an autosomal recessive genetic disease (formerly known as P450 oxidoreductase deficiency) with clinical phenotypes of ambiguous genitalia, congenital adrenal hyperplasia, skeletal malformation, Antley-Bixler syndrome (ABS), and polycystic ovary syndrome Miller’s group have evaluated the impact of polymorphic variants of POR on the catalytic activity of CYP1A2 and CYP2C19 in vitro. In order to maintain the physiological catalytic activity of POR and CYP as practically as possible, we have selected the following six full-length POR missense mutations . These mutations in POR  are associated with Antley-Bixler Syndrome, and A287P was found to be the most common POR mutation in the European population. Missense mutation K49N is located in the amino-terminal tail of POR which is responsible for anchoring POR into the endoplasmic reticulum (or plasma membrane) and is important for proper electro transfer function, similarly, S244C is located within the hinge close to FAD and FMN domains of POR. Therefore, we expressed these six POR missense mutations in baculovirus/Sf9 insect cell system , and then co-expressed with wild-type human CYP3A4 and CYP2B6 respectively, to investigate the influence of these POR genetic variations on the metabolic activities of CYPs .The present study was carried out to reveal the potential role of POR genetic variations on the CYP3A4 and CYP2B6 mediated metabolic activities and to help set up a database about the influence of POR genetic polymorphisms as a biomarker to predict the POR-involved metabolic activities of clinical drugs 2, Tris–HCl and other chemicals and solvents were analytical reagents obtained from Sinopharm Chemical Regent Co.  or chromatographic grade obtained from Tedia, Co. .Polymerase chain reaction (PCR) primers were synthesized by Sunny Biotechnology . Restriction endonucleases, DNA molecular marker, and T4 ligase were obtained from MBI Fermentas . Site-directed Mutagenesis Kit , Cellfectin reagent, pFastBac1 vector, DH10Bac-competent cells, Grace’s medium and fetal bovine serum were purchased from Invitrogen . Spodoptera frugiperda Sf9 insect cells were obtained from Yangshengtang Company . The pFastBAc-3A4 and 2B6 plasmids were kindly provided by Dr. Su Zeng (Zhejiang University). The primary anti- histidine antibody and Peroxidase-conjugated goat anti-mouse secondary antibody were purchased from Abmart  and Jackson ImmunoResearch Laboratories, Inc  respectively. Testosterone and bupropion  were purchased from National Institutes for Food and Drug Control . Ammonium acetate, acetic acid, formic acid, acetonitrile, MgClPOR*25; A115V, POR*11; Y181D, POR*8; S244C; A287P, POR*5; G413S) were classified according to the Human CYPallele nomenclature committee .Site-directed mutagenesis was performed using Site-directed Mutagenesis Kit  according to manufacturer’s instructions. The template plasmid with cDNAs encoding wild-type POR tagged with histidine (His6) has been previously cloned into pFastBac1 in our laboratory. Primers used in site-directed mutagenesis are listed in 8 pfu/ml. Sf9 insect cell were obtained from Yangshengtang Company  and maintained at 27°C in Grace’s medium containing 10% FBS. Sf9 cells were co-infected by CYP3A4 or CYP2B6 with POR recombinant baculovirus particles, and then Hemin stock solution  was added to the culture medium for a final concentration of 2 µg/ml. After incubation for 72 h, the infected Sf9 cells were harvested, washed with ice-cold phosphate balanced solution  and re-suspended in the lysis buffer . The microsomes were prepared by centrifugation at 9000×g for 10 min at 4°C after sonication of cells to obtain the supernatant which was then stored at −80°C until use.The Bac-to-Bac baculovirus expression system was used for the expression of CYP3A4, CYP2B6 and POR enzymes. All steps for the production of recombinant proteins were carried out as described previously Tm Kit . The primers ATGATCAACATGGGAGACTCCCACGTGG (forward) and CTAGCTCCACACGTCCAGGGAGTAGC (reverse) were used to detect the viral DNA.The recombinant viral titers of CYP3A4, CYP2B6 and wild-type or mutant PORs were identified using real-time PCR, it has been previously demonstrated that there exists good agreement between DNA copy number and virus titers −1Sf9 cells were harvested, and washed twice with cold PBS. Cell pellets were lysed by sonication in the presence of protease inhibitors and the total protein concentration was determined by BCA protein assay kit. Twenty microgram of each protein sample was resolved by 8% SDS-PAGE and electroblotted onto nitrocellulose membranes . The membranes were blocked for 1 h at room temperature in PBS containing 5% skim milk plus 0.1% Tween-20 (PBST) and was incubated overnight at 4°C with monoclonal antibody against histidine as primary antibody (1∶3000 dilution), followed by incubation with a secondary antibody  for 1 h at room temperature. The blots were detected with Immobilon Western chemiluminescent HRP (horseradish peroxidase) substrate (Millipore) by a lumino image analyzer, LAS-1000 plus (FujiFilm) according to the manufacturer's instructions. The amount of POR in each microsomal fraction was detected by western blotting, and the quantification of the immunoblots was performed by measuring the scanned band intensities using Quantity One-4.6.2 Software. POR activity assay was performed by the reduction of cytochrome c in the presence of wild-type POR and mutants POR respectively 2, NADPH generating system resulting in a final concentration of 1 unit isocitrate dehydrogenase/ml and 5 mM isocitrate. Concentrations of testosterone or bupropion in the incubation mixtures ranged from 20 µM to 400 or 20 µM to 800 µM for CYP3A4-PORs or CYP2B6-PORs. The reaction mixture was started by addition of NADPH/NADP+ at final concentration of 1 mM and terminated after 60 min by the addition of two volumes of ice-cold acetonitrile. After incubation, the reaction mixture was centrifuged at 16,000×g for 20 min to obtain the supernatant and pellet protein. The supernatant (20 µL) was then assayed directly by using HPLC (Agilent Technologies 1200 series). All chromatographic separations were obtained using a Dikma Technologies’s, Diamonsil C18 column . For detection of testosterone, it was monitored at 244 nm by C18 column with acetonitrile-water  as mobile phase, at a flow rate of 1.0 ml/min 2PO4  40:60 (v/v) as mobile phase, at a flow rate of 1.0 ml/min as reported elsewhere Microsomes of CYP3A4 and CYP2B6 co-expressed with wild-type or mutant PORs were determined for their metabolic ability for testosterone and bupropion respectively. Each sample was pre-incubated for 5 min at 37°C in a total volume of assay buffer (100 µL), which included 50 pmol of CYP3A4-POR or CYP2B6-POR microsomes, 100 mM Tris-HCl (pH7.4), 15 mM MgClEach experiment was performed at least three times and all data has been presented as mean ± S.D. The Vmax and apparent Km values were determined using nonlinear regression analysis of Michaelis-Menten model  Statistical analysis of data was carried out using a one-way ANOVA followed by Holm-Sidak pairwise multiple comparison test . A probability value of less than 0.01 and 0.05 (*p<0.01 and *p<0.05) was accepted as a significant difference.In order to better understand the effects of polymorphic variants of POR on CYPs, six different mutants POR were heterologously co-expressed with CYP2B6 and CYP3A4 in sf9 insect cells and were compared with activity of wild-type. The amount of POR in each microsomal fractions were quantitated by western blotting according to the published method in vitro using reduction of Cytochrome c system, as described in method. The changes in the activity of each POR  in cells expressing PORs alone or co-expressing with CYP3A4 (or CYP2B6) are summarized in In order to understand the influences of mutations on POR activity, the specific enzymatic activities of six POR mutants were determined On the other hand, the efficiency of each POR activity  in cells co-expressing PORs with CYP3A4 (or CYP2B6) was found to be similar to the cells expressing PORs alone , but actWe further sought to determine whether the six polymorphic variants of POR can alter CYPs-mediated metabolic variations, for which CYP3A4 and CYP2B6 were co-expressed with six POR-mutants (respectively) in sf9 insect cells. Afterward, the metabolites of testosterone (as a specific substrate for CYP3A4) and bupropion (as a specific substrate for CYP2B6) were determined to establish the changes in the activities of CYP3A4 and CYP2B6. The metabolic products were determined by HPLC with their authentic standards . int) when compared with wild-type. In particular, CYP3A4-POR (G413S) exhibited the highest changes in the Km and Vmax (1.3-folds) values compared to wild-type, and increased the catalytic efficiency (CLint) up to 65% of wild-type. Moreover, CLint values of CYP3A4-POR (K49N) and CYP3A4-POR (A115V) were calculated to increase up to 31% and 36% of wild-type. Additionally, mutant S244C of POR also increased the activity of CYP3A4, but it was much less than that of K49N, A115V and G413S, as shown in On contrary, the remaining three variants, K49N, A115V and G413S of POR showed a significant increase in the catalytic efficiencies of CYP3A4-POR, which was judged by the ratios of Vmax/Km  with bupropion (as a specific substrate for CYP2B6), we found that the influences of POR genetic mutations on catalytic activities of CYP2B6 were different as compared to that of CYP3A4, as shown in With respect to A115V and G413S, the activities of CYP2B6 were not affected significantly by co-expressing with these two mutants , while tPharmacogenetics has been used recently to establish correlations between genetic polymorphisms and variations in drug efficacy, which might contribute to the development of personalized medicine in the near future. Additionally, an individual’s genetic makeup and genetic polymorphism may lead to optimization of therapeutic strategies resulting in maximum therapeutic efficiency and minimal adverse drug reactions Cytochrome P450 oxidoreductase (POR) is known to play a critical role in delivering electrons to all microsomal P450 enzymes and many of the redox system E.coli, suggesting that the full-length POR variants may be more suitable for evaluating the human POR-variants The human POR variants were recently expressed by using truncated cDNAs in bacterial system (prokaryocytes) to evaluate the effects of the POR on drug metabolism. However, in this system the expressed POR proteins were found to lack many amino acid residues  In order to assess the activities of POR variants, each POR was expressed independently or co-expressed with CYP3A4 or CYP2B6 in sf9 cells. The activities of POR variants were determined by measuring reduction of Cyt c. Interestingly, the mutants Y181D and A287P in POR strongly reduced the activity to approximately 20% of wild-type, while S244C and G413S enhanced the POR activity up to 13% and 77% respectively compared with wild-type , suggestAdditionally, we determined the Km and Vmax values for two specific substrates  by CYP3A4-POR and CYP2B6-POR. The activity of CYP3A4 was completely inhibited after co-expressing with Y181D and A287P when compared with the wild-type , this reVmax/Km values were increased up to 31% of wild-type, while it decreased in CYP2B6-POR (K49N) to 74% of wild-type  for ild-type . Furtherild-type . S244C min vitro or in vivo drug clearance tests. In summary, we suggested that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 for the prescribed medication to obtain maximum therapeutic efficiency and minimal adverse drug reactions. Therefore, our present study would be helpful to set up a database about the influence of POR genetic polymorphisms as a biomarker to predict the POR-involved metabolic activity of clinical drugs.To date, more than 50 microsomal P450 enzymes and 40 mutants POR have been found in human Figure S1Immunoblot analysis of wild type and six mutant POR microsomal proteins in Sf9 cells. (A) The position of POR proteins was detected at 78 kDa by Western blot. N: negative. W: wild type. (B) The relative expression levels of wild type and mutants POR in Sf9cells were quantified with Quantity One Software. The results are indicated as mean ± S.D. of three independent experiments.(TIF)Click here for additional data file.Figure S2Immunoblot analysis of CYP3A4 microsomal proteins co-expressed with wild type or six mutants POR in Sf9 cells. (A) The position of CYP3A4 proteins were detected at 54 kDa by Western blot after infections with wild type or mutants POR, as described in Methods. (-): negative. WT: wild type. (B) The relative expression levels of CYP3A4 proteins in POR-infected Sf9 cells were quantified with Quantity One Software. The results are indicated as mean ± S.D. of three independent experiments.(TIF)Click here for additional data file.Figure S3Immunoblot analysis of CYP2B6 microsomal proteins co-expressed with wild type or six mutants PORsSf9 cells. (A) The position of CYP2B6 proteins was detected at 56 kDa by Western blot after infections with wild type or mutants POR, as described in Methods. (-): negative. WT: wild type. (B) The relative expression levels of CYP2B6 proteins in POR-infected Sf9 cells were quantified with Quantity One Software. The results are indicated as mean ± S.D. of three independent experiments.(TIF)Click here for additional data file.Figure S4CO-difference spectrum of the expressed CYP3A4 (A) and CYP2B6 (B). Content was determined from the carbon monoxide difference spectrum and the molar absorption coefficient for cytochrome P450.(TIF)Click here for additional data file.Table S1PCR primers used for site-directed mutagenesis of POR cDNAs.(DOCX)Click here for additional data file.Table S2Real-time PCR titration results for CYP3A4, CYP2B6 and wild type or mutant PORs.(DOCX)Click here for additional data file.


RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB) repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation. This localization pattern depends on the presence of an intact, ubiquitin-binding Zinc finger domain. Using a biochemical approach, we show that RAD18 directly binds to ubiquitylated H2A and several other unknown ubiquitylated chromatin components. This interaction also depends on the RAD18 Zinc finger, and increases upon the induction of DSBs by γ-irradiation. Intriguingly, RAD18 does not always colocalize with regions that show enhanced H2A ubiquitylation. In human female primary fibroblasts, where one of the two X chromosomes is inactivated to equalize X-chromosomal gene expression between male (XY) and female (XX) cells, this inactive X is enriched for ubiquitylated H2A, but only rarely accumulates RAD18. This indicates that the binding of RAD18 to ubiquitylated H2A is context-dependent. Regarding the functional relevance of RAD18 localization at DSBs, we found that RAD18 is required for recruitment of RAD9, one of the components of the 9-1-1 checkpoint complex, to these sites. Recruitment of RAD9 requires the functions of the RING and Zinc finger domains of RAD18. Together, our data indicate that association of RAD18 with DSBs through ubiquitylated H2A and other ubiquitylated chromatin components allows recruitment of RAD9, which may function directly in DSB repair, independent of downstream activation of the checkpoint kinases CHK1 and CHK2. RAD18 knockout cells are sensitive to UVC light exposure in vitroMammalian cells require the E3 ubiquitin ligase RAD18 for survival after the induction of various types of DNA damage. S. cerevisiae and human cells have shown that RAD18 interacts directly with RPA S. cerevisiae and mammalian cells In addition to the RDB pathway, RAD18 also functions in DSB repair. DSBs may arise from exogenous factors such as ionizing radiation. In addition, DSBs can arise when the replication fork collapses during S phase. Two distinct DSB repair pathways have been identified in mammalian cells; non-homologous end-joining (NHEJ), and homologous recombination (HR). NHEJ is an error-prone form of DSB repair, in which the two ends of the broken DNA are processed for direct ligation. This mechanism is thought to be operative mainly during the G1 phase. In contrast, HR is an error-free mechanism, in which a homologous sequence of the sister chromatid is used as a template to process repair in S and G2 phases. All HR pathways are initiated by 5′-3′ degradation of one strand at both sides of the break; the so-called DNA-end resection, generating stretches of ssDNA, that are subsequently coated by the ssDNA binding protein complex RPA , and to mediate its ubiquitylation The chromatin surrounding DSBs undergoes various modifications. One of the first modifications is phosphorylation of histone H2A variant H2AX (γH2AX) Herein, we aim to determine how RAD18 is recruited to DSBs, and the downstream effects of RAD18 functions at DSBs. We report that RAD18 can interact directly with ubiquitylated H2A and other ubiquitylated chromatin components via its Zinc finger domain, and this binding increases upon induction of DSBs. Furthermore, RAD18 colocalizes with ubiquitylated H2A throughout the cell cycle. Finally, we reveal that RAD18 facilitates the accumulation of RAD9 at DSBs.RAD18 contains several distinct functional domains; a C3HC4 type RING finger, a C2HC type Zinc finger, a SAP domain, and a HR6A/B binding domain (HR6BD)  [14]. Wein vivo. However, it cannot be excluded that the autoubiquitylation site changes when the preferred lysine is mutated.In the analysis of the immunoblots, HeLa cells stably expressing YFP-RAD18 at a level similar to endogenous RAD18  [28] werSaccharomyces cerevisiae, Rad6 binds to Rad18 via the C-terminal domain of Rad18, as shown in a yeast two-hybrid assay in vitro assay in vivo. Indeed, a double mutant RAD18 (C28F/A357R) completely failed to co-precipitate HR6A/B, confirming that HR6A/B interacts with RAD18 via both the HR6BD and the RING finger domains in vivo  Having established that the Zinc finger domain of RAD18 is required for the formation of RAD18 foci throughout the cell cycle and upon irradiation, we next investigated which proteins are required for RAD18 recruitment to chromatin.In order to examine whether RPA is required for RAD18 accumulation at sites of γ-irradiation-induced DNA damage, RPA2 was depleted using siRNA, and foci formation of RAD18 was analyzed. Downregulation of RPA2 expression  also redFollowing induction of DSBs, the surrounding chromatin at the break sites undergoes various modifications. One of the first modifications is phosphorylation of histone H2A variant H2AX (γH2AX) by ATM (serine/threonine protein kinase ataxia telangiectasia mutated) The immunocytochemical analyses of conjugated ubiquitin in relation to the localization of RAD18 in HeLa cells indicate that RAD18 localization to chromatin is tightly correlated to the presence of ubiquitylated H2A and other ubiquitylated chromatin components . To examNext, to analyze whether RAD18 always colocalizes with regions that show enhanced H2A ubiquitylation, we analyzed human female primary fibroblast cultures. In these cells, dosage compensation has resulted in the inactivation of one of the two X chromosomes, and the inactivated chromosome, forming the Barr body, is known to be enriched for ubiquitylated H2A and trimethylation of histone H3 at lysine 27 (H3K27me3). Immunocytochemical analyses of ubiquitylated H2A, conjugated ubiquitins, H3K27me3, and RAD18 signals in these cells revealed that the Barr body, visible by dense DAPI staining, and H3K27me3 (data not shown) was mostly not enriched for RAD18, even if uH2A staining was enhanced in this region. In a small percentage of the nuclei (6%), RAD18 enrichment was detected at the Barr body . SometimRAD18 knockdown HeLa cell lines . Taken together, these data suggest that recruitment of RAD9 to IR-induced DSBs requires the Zinc finger domain of RAD18 and its E3 ligase activity. To analyze this further, we studied physical interactions between RAD18 and components of the 9-1-1 complex by the yeast two-hybrid assay in the absence and presence of DNA damage . In accordance with recent data concerning yeast RAD18 and the 9-1-1 complex Rad18 knockdown cells  who found that the Zinc finger domain of RAD18 is required for focal localization of RAD18 at damaged sites in cells irradiated with an UVA or UVC laser. In addition, RAD18 carrying a mutation in the Zinc finger domain could restore Polη foci formation at sites of stalled replication, whereas a SAP domain mutant could not RPA is a single-stranded DNA (ssDNA) binding protein, essential for both DNA replication and recombination RPA knockdown cells indicates that RPA accumulation and function are somehow linked to this branch of the DDR pathway.Our observation that not only the accumulation of RAD18, but also that of ubiquitylated chromatin components depends on RPA is surprising. Ubiquitylation of chromatin in response to DSBs requires the ubiquitin ligases RNF8 and RNF168 In the present study, we found a physical and specific interaction between RAD18 and ubiquitylated H2A via its Zinc finger. In addition, certain other ubiquitylated chromatin components may also be bound by RAD18. The distribution pattern of conjugated ubiquitin on chromatin in the nucleus during the cell cycle was identical to that of RAD18, indicating that chromatin association of RAD18 primarily occurs through ubiquitin-binding. Intriguingly, immunocytochemical analyses in human female primary fibroblast cells revealed that the Barr body, known to be marked with uH2A, was mostly not enriched for RAD18, even if uH2A and FK2 staining was enhanced in this region. These data indicate that the interaction of RAD18 with ubiquitylated chromatin components including uH2A is related to DNA damage, and possibly regulated. Such regulation might occur via phosphorylation of RAD18 by ATM kinase, similar to what occurs for many other proteins involved in the DNA damage response pathway.In vitro ubiquitylation of RAD18 depends on HR6A/B in vivo , also showed that the role of RAD18 in HR requires the Zinc finger and RING finger domains of RAD18, although their data indicated that the E3 ligase activity of RAD18 is not required to promote HR Earlier reports have also shown association of human RAD9 with chromatin after DNA damage Taken together, our data suggest that RPA is required for RAD18 localisation at DSBs. This most likely occurs through indirect recruitment of RNF8/RNF168 to DSBs, and subsequent ubiquitylation of H2A and other chromatin components. The RAD18 zinc finger domain binds to these ubiquitin moieties, resulting in efficient RAD18 recruitment, and this facilitates RAD9 accumulation via the ubiquitin ligase activity of RAD18. The role of RAD18 in stimulating HR may thus be mediated in part through direct actions of RAD9 interacting with RAD51 and/or RPA.DpnI (Bio England) for 2 hours at 37°C. XL1-blue (Strategene) was transformed with 2 µl of the DpnI digested PCR products. Plasmids containing appropriate mutants were digested with EcoRI and BamHI and sub-cloned into pEYFP-C1 (Clontech) and mCherry-C1 (Clontech). pEGFP-RAD9 construct was kindly provided by Daniel Warmerdam Design of the YFP-RAD18 construct containing four silent mutations, and generation of the HeLa cell line stably expressing YFP-RAD18 were described elsewhere RAD18, PCNA, RAD9, HUS1 and RAD1, and mouse Hr6a and Hr6b genes were amplified using primer sets  were annealed  and sub-cloned into pSuper-puro vector (OligoEngine). To generate HeLa cells stably expressing pSuper-shRAD18, cells were transfected with 2 µg of the pSuper-shRAD18 plasmid using FuGene6 (Roche). Cells were cultured for 7 days after transfection with medium containing 0.1 mM of puromycine. Surviving colonies were selected, and RT-PCR and immunoblot analysis were used to further select cell lines that showed downregulation of RAD18. As a control, the empty pSuper-puro vector was transfected followed by the same selection procedure. In order to transfect mutant RAD18 in the RAD18 knockdown cell line, five silent mutations were introduced in the RAD18 gene in the region that is targeted by pSuper-shRAD18 2 in air. Female human primary fibroblast was cultured at 37°C in DMEM/F12 (GIBCO) supplemented with 15% v/v fetal calf serum, 1% non essential amino acid (GIBCO), 0.1 mM β-mercaptoethanol, and penicillin, under 5% CO2 in air.HeLa cells were cultured at 37°C in DMEM/F12 (GIBCO) supplemented with 5% v/v fetal calf serum, streptomycin sulfate and penicillin, under 5% COin vitroAll four putative ubiquitylation sites  were mutated to an alanine residue (Δubi). In the RING finger, C28 was mutated to phenylalanine (C28F), to abolish the E3 ligase activity For primary antibodies, we used mouse monoclonal antibodies anti-GAPDH (Chemicon), anti-GFP (Roche), anti-RPA2 (Thermo Scientific), anti-FK2 , anti-PCNA PC10 (Abcam), anti-ubiquitylated histone H2A , and rabbit polyclonal antibodies anti-RAD18 5 cells were plated into six-well culture dishes. After 24 hours, cells were transfected with siRNA and incubated for 72 hours. Each transfection mixture contained 9 µl HiPerFect Transfection Reagent (Qiagen) and 2 µM siRNA (Ambion) in 400 µl serum-free DMEM/F12. siRNA28198 (si-endoRAD18) (Ambion) was used to downregulate only endogenous RAD18 but not YFP-RAD18 or mutant YFP-RAD18. siRPA1 (Ambion) Portions of 2×10TCTGTATGCATGGGACAGGA, reverse/TCAGGTTCCAATTCCTCTGG. PCR of β-actin mRNA was included in each reaction and used to normalize the data. Three independent experiments were performed and each real-time PCR was performed in duplicate. All –RT reactions were negative.For real-time RT-PCR, RNA was prepared by Trizol from HeLa cells stably expressing shRAD18 or non-targeting shRNA, DNase-treated and reverse transcribed using random hexamer primers and Superscript II reverse transcriptase (Invitrogen). PCR was carried out with the iQ SYBR green PCR mastermix (Applied Biosystems) in the DNA engine Opticon 2 real-time PCR detection system (Bio-Rad) with RAD18 primer; forward/137Cs source (0.71 Gy/minutes).To induce global ionizing damage, cultured cells were irradiated with γ-rays from a 2. For immunoblot analysis, cells were cultured for another 6 hours and whole-cell extracts were prepared.To induce global UVC damage, cultured cells were irradiated with a Philips TUV lamp (254 nm) at a dose of 20 J/m2, 0.5 mM EDTA, 0.2% Nonidet P-40, 10% (v/v) glycerol, 0.5 mM dithiothreitol, and protease inhibitors (Roche)). The cell suspension was left on ice for 1 h, sonicated 5 times for 5 sec. Subsequently the lysate was treated with 0.125 U/µl of benzonase nuclease (Novagen) for 2 h, and centrifuged for 30 min (full speed at 4°C). The efficiency of nuclease was checked by DNA precipitation. The cell lysate was diluted with Lysis buffer 2  glycerol, 0.5 mM dithiothreitol, and protease inhibitors (Roche)) to decrease the concentration of NaCl to 150 mM. The cell lysate was pre-cleared with 20 µl of protein A-agarose beads  for 2 hours at 4°C. To cross-link antibodies with beads, antibodies (1 µg) were first incubated with 20 µl of beads for 2 hours at 4°C. The beads were washed three times with lysis buffer and subsequently incubated with borate buffer pH 8.0 containing 20 µM dimethyl pimelimidate (Sigma) for 1 hour at RT. The beads were washed once with ethanol amine pH 8.0 and incubated for 1 hour at RT. The beads were washed three times with lysis buffer and incubated with lysis buffer containing 5% BSA for 2 hours at 4°C. Subsequently, the beads were used for immunoprecipitation analysis. Each immunoprecipitation analysis contained 2 mg of lysate and 25 µl of protein A-agarose, and the mixture was incubated overnight at 4°C. After washing three times with 500 µl of Lysis buffer, bound proteins were analyzed by SDS–PAGE and immunoblots.HeLa cells expressing either wild type or mutant YFP-RAD18 were lysed in 2.0 ml Lysis buffer 1 , and sonicated. After 5 minutes incubation at 100°C, the cell extracts were separated by SDS 10% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were incubated with antibodies to analyze expression of target proteins. The expression was detected using enhanced chemiluminescence (PerkinElmer). The target proteins detected using IRDye680 or IRDye800 were quantified with Odyssey (Li-cor).+ (PBS containing 0.5% w/v BSA and 0.15% w/v glycine) for 30 minutes, incubated with primary antibody for 2 hours, and washed with 0.2% Triton X-100 in PBS. Subsequently, cells were incubated with secondary antibody for 1 hour, washed with 0.2% Triton X-100 in PBS. Coverslips were placed on a slide, mounted with Prolong Gold reagent (Invitrogen).Cells were cultured on 24 mm round coverslips, washed with PBS and fixed in 2% w/v paraformaldehyde in PBS for 15 minutes. To detect ubiquitylation of histone H2A, cells were pretreated with 0.2% v/v Triton X-100 in PBS for 30 seconds before the fixation step. Cells were permeabilized for 20 minutes with 0.2% v/v Triton X-100 in PBS. Cells were blocked with PBS2. YFP-tagged proteins were detected by exciting YFP with a 488 nm Argon gas laser and monitoring YFP emission through a 500–550 band-pass filter. mCherry-tagged proteins were detected by exciting mCherry with a 543 nm helium neon laser and monitoring mCherry emission through a long-pass 560 filter. To minimize the effect of photo-bleaching, images were taken with 10 µW for a 488 nm laser, and with 20 µW for a 543 nm laser. For time-lapse experiments, cell images of 6 confocal planes at 1.5 µm intervals were taken every 20 minutes for 27 hours. All images were captured with a line average of 2. Time-lapse images were analysed using the ImageJ software .Images of living cells expressing YFP-tagged and mCherry-tagged proteins were obtained using a Zeiss LSM510NLO microscope (Carl Zeiss) with a 63×/1.40 NA oil immersion lens. Cells were maintained at 37°C in a mixture of air with 5% CO2 (40×40 pixels), and the output of laser power was set at 75 mW at pixel-dwell time 1.6 µs with 5 iterations. Two images were taken before MPL irradiation and monitored at 10 seconds intervals for 10 minutes immediately after MPL irradiation. All images were captured with a line average of 1.A Coherent Mira mode-locked Ti:Sapphire laser  (Coherent) connected to a Zeiss LSM510NLO confocal microscope was used at 800 nm with a pulse length of 200 fs and repetition rate of 76 MHz. For local DNA damage induction, an area of irradiation was set at 4 µmSaccharomyces cerevisiae AH109 (Clonetech) were cultured in YPD medium (1% (w/v) yeast extract (Difco), 2% peptone (w/v) 2% glucose (w/v)) at 30°C. When the density of the medium was 1.0, cells were harvested and washed twice with distilled water, mixed with transformation buffer , 5 µl 10 mM salmon sperm DNA (Clontech), 50 µl H2O, 4 µg of pGADT7 vector carrying target DNA with Gal4 activation domain, and 4 µg of pGBKT7 carrying target DNA with Gal4 binding domain), incubated 30 min at 30°C, and subsequently 30 min at 42°C. The transformation buffer was discarded and 10% of transformed cells were spread onto the selection drop medium SD-L-W (Clontech). Interactions were determined by spot assay on SD-L-W-H (Clontech) and SD-L-W-H-A (Clontech) indicator plates.Figure S1Knockdown of RAD18 and functional analyses of RAD18 mutants. (A) Transient downregulation of RAD18 with siRNA against Rad18 in Hela cells (si-RAD18) stably expressing GFP-RAD9 and transiently co-expressing mCherry-PCNA. (B) The level of RAD18 mRNA and protein expression in three different HeLa cell lines stably expressing shRNA targeting RAD18. The control level, in HeLa cells stably expressing non-targeting shRNA, was set at 1.0. (C) Expression of endogenous RAD18 was detected by immunostaining using anti-RAD18 in control and RAD18 knockdown cell lines (shRAD18). Cells were irradiated with IR (5 Gy), and fixed after 1 hour. (D) Analyses of RAD18 ubiquitylation. Endogenous RAD18 was transiently knocked down in HeLa cells stably expressing YFP-RAD18 or transiently expressing YFP-RAD18Δubi. Cells were lysed (input) and immunoprecipitation with YFP antibody (IP αYFP) was performed. The expression levels of RAD18 and HR6A/B in the lysate are shown as input. Immunoprecipitated RAD18 and co-immunoprecipitated HR6A/B were detected on immunoblots (IP αYFP). (E) Endogenous RAD18 was transiently knocked down (si-endoRAD18) in HeLa cells transiently expressing YFP-RAD18 mutated in putative auto-ubiquitylation sites. Auto-ubiquitylation of YFP-RAD18 was analyzed on immunoblots. GAPDH was used as a loading control. (F) Endogenous RAD18 was transiently knocked down (si-endoRAD18) in HeLa cells stably expressing YFP-RAD18 or transiently expressing YFP-mutant RAD18. Cells were irradiated with 20 J/m2 UVC, and whole-cell extracts were prepared 8 h after irradiation. Expression levels of mono-ubiquitylated PCNA were analyzed on immunoblots using αPCNA. Arrowheads point at mono-ubiquitylated PCNA (ubi-PCNA). The panels show the results of a representative experiment, where three independent experiments yielded similar results. (G) Cell lysates from wild type HeLa cells and HeLa cells stably expressing YFP-RAD18 were treated with or without benzonase nuclease for 2 h on ice. In the presence of benzonase, DNA was absent from the cell lysates. M in the figure indicates DNA ladder.(TIF)Click here for additional data file.Figure S2Subnuclear localization of wild type and mutant YFP-RAD18 during the cell cycle and after irradiation. Confocal images of living HeLa cells expressing wild-type or mutant YFP-RAD18 in G1, S, and late G2 phases, and after irradiation with IR (5 Gy). Endogenous RAD18 was downregulated by siRNA (si-endoRAD18). RAD18 carrying mutations in its Zinc finger  and a deletion of the Zinc finger (ΔZINC) showed no cell cycle specific localization. In contrast, RAD18 carrying either mutations of the RING finger  or SAP domain  showed a localization pattern identical to that of wild-type RAD18 (WT).(TIF)Click here for additional data file.Figure S3No direct interaction between RAD18 and components of the 9-1-1 complex. Yeast two-hybrid assay. Yeast carrying both pGADT7-target DNA and pGBKT7-target DNA were spotted on SD-L-W plates to confirm the yeast transformation. The interaction was confirmed by growth on selective medium plates, SD-L-W+X-Gal, SD-L-W-H and SD-L-W-H-A. The protein expression of transformed plasmids was examined on immunoblots by different antibodies as indicated. pGADT7 vector contains a HA-epitope tag, and the pGBKT7 vector contains a c-myc epitope tag. Three independent experiments were performed and representative results are shown. A) Yeast two hybrid assay between RAD18 and all 9-1-1 components. Various types of DNA damage were induced by irradiation with 50 Gy, or growth on the selective medium plates containing 25 mM HU, 0.05% MMS, or 8 µM CPT. Different concentrations of HU , MMS , CPT  were examined and showed similar results (data not shown). B) Yeast two hybrid assay between the 9-1-1 components. C) Yeast two hybrid assay between RAD18, and HR6A, HR6B or PCNA.(TIF)Click here for additional data file.Figure S4Normal CHK1 and CHK2 phosphorylation in RAD18 knockdown Hela cells. HeLa cells stably expressing non-targeting shRNA or shRNA targeting RAD18 were exposed either with UV at 20 J/m2 or IR at 10 Gy. Prior to irradiation, and after certain time points indicated in the figure, phosphorylation of CHK1 at Ser 354 and CHK2 at Tyr 68 was analyzed on immunoblots. GAPDH was used as a loading control.(TIF)Click here for additional data file.Movie S1Analyses of wild type RAD18 localization during the cell cycle. Time lapse images of nuclei expressing wild type YFP-RAD18. Images were captured every 30 min for 27 hours, a single nucleus was selected, aligned, and 5 frames of the images are shown per second. Endogenous RAD18 was downregulated by siRNA. Analyses began in S phase, and the cell went through G2, M and G1. The growth rates of wild type and mutant cells were not different (data not shown).(AVI)Click here for additional data file.Movie S2Analyses of mutant RAD18 localization during G1, S and G2 phase. Time lapse images of nuclei expressing YFP-RAD18 carrying a mutation in its Zinc finger (D221A). Images were captured every 30 min for 27 hours, a single nucleus was selected, aligned, and 5 frames of the images are shown per second. Endogenous RAD18 was downregulated by siRNA. Analyses started in G1 phase, and the cell went through S, and most likely G2. The cell cycle phases of mutant YFP-D221A were inferred from the time passed since the previous M-phase or the time span until the next M-phase, and the duration of G1, S, G2 and M phase in wild type cells. The growth rates of wild type and mutant cells were not different (data not shown).(AVI)Click here for additional data file.Movie S3Analyses of mutant RAD18 localization during M, G1 and S phase. Time lapse images of nuclei expressing YFP-RAD18 carrying a mutation in its Zinc finger (D221A). Images were captured every 30 min for 27 hours, a single nucleus was selected, aligned, and 5 frames of the images are shown per second. Endogenous RAD18 was downregulated by siRNA. Analyses began in G2 phase and the cell went through, M, G1, and most likely S. The cell cycle phases of mutant YFP-D221A were inferred from the time passed since the previous M-phase or the time span until the next M-phase, and the duration of G1, S, G2 and M phase in wild type cells. The growth rates of wild type and mutant cells were not different (data not shown).(AVI)Click here for additional data file.Table S1Designed primers to create mutant RAD18. Mutated amino acid codons are shown in red, and the introduced mutations are shown in bold italics(DOC)Click here for additional data file.Table S2Primers used to generate Y2H vectors. Restriction enzyme sites used for subcloning are underlined.(DOC)Click here for additional data file.Table S3Primers used to generate a vector carrying the shRAD18 sequence.(DOC)Click here for additional data file.Table S4siRNAs used in this study.(DOC)Click here for additional data file.


These studies provide new insight into the biology of DENV pathogenesis and the requirements for antibodies to treat lethal DENV disease.Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are life-threatening complications following infection with one of the four serotypes of dengue virus (DENV). At present, no vaccine or antiviral therapies are available against dengue. Here, we characterized a panel of eight human or mouse-human chimeric monoclonal antibodies (MAbs) and their modified variants lacking effector function and dissected the mechanism by which some protect against antibody-enhanced lethal DENV infection. We found that neutralizing modified MAbs that recognize the fusion loop or the A strand epitopes on domains II and III of the envelope protein, respectively, act therapeutically by competing with and/or displacing enhancing antibodies. By analyzing these relationships, we developed a novel  in vitro and in vivo. In this study, we studied a larger panel of modified MAbs that recognize different regions of the DENV envelope protein. While all modified MAbs acted therapeutically to prevent a lethal, virus-only DENV infection, only certain MAbs effectively protected mice following an antibody-enhanced lethal infection. We determined that therapeutically effective MAbs following an ADE infection worked by competing for binding of enhancing antibodies on the DENV virion. Based on this, we designed an in vitro suppression-of-enhancement assay that predicted the ability of modified MAbs to act therapeutically in vivo.The four dengue virus serotypes (DENV1-4) cause the most prevalent mosquito-transmitted viral disease globally, infecting 50–100 million people annually in tropical and sub-tropical regions worldwide, yet no vaccine or therapy has been licensed to prevent or treat dengue. The greatest risk factor for severe dengue disease is a previous infection with a different serotype, which is thought to be due in part to a phenomenon known as antibody-dependent enhancement (ADE) whereby anti-DENV antibodies from a prior infection augment DENV infection of target Fcg receptor (FcgR)-expressing cells. We previously developed a mouse model that demonstrates antibody-enhanced lethal DENV disease and showed that genetically-modified antibodies incapable of interacting with the FcgR eliminate ADE  Aedes aegypti and Ae. albopictus mosquitoes and are endemic predominantly in tropical and sub-tropical regions of the world The four serotypes of dengue virus (DENV) are transmitted by Flavivirus genus and is closely related to other medically important arboviruses including West Nile (WNV), Japanese encephalitis, tick-borne encephalitis, and yellow fever viruses DENV is a member of the in vivo, even when administered one or two days after infection. Here, we evaluated the therapeutic activity of a larger panel of MAbs targeting different epitopes on the E protein following both a virus-only as well as an antibody-enhanced lethal infection. We determined that the two most potent therapeutic MAbs acted by competitively displacing either fusion-loop specific MAbs or enhancing polyclonal serum antibodies targeting a proximal epitope. Using this information, we designed a novel suppression-of-enhancement assay in human FcγRIIA-expressing K562 cells that predicts the ability of modified MAbs to act therapeutically against antibody-enhanced disease in vivo. Our observations provide new insight into the mechanism by which therapeutic MAbs prevent an antibody-enhanced lethal DENV infection.Epidemiological analysis has established that a previous DENV infection is the greatest risk factor for the development of severe disease Severe forms of DENV infection, including DHF/DSS, can be fatal, as no specific antiviral therapy is currently available. As such, we extended previous observations of the prophylactic and therapeutic efficacy of the EDII fusion loop-specific MAb E60 6 PFU) of DENV2 D2S10 and 24 hours later, administered 20 µg of individual genetically modified MAbs lacking the ability to bind FcγR or C1q. Notably, all of the modified MAbs tested prevented development of overt disease and protected against death in this model . We subsequently assessed whether these MAbs also protected against antibody-enhanced lethal DENV infection. Anti-DENV1 serum was administered 24 hours prior to a sub-lethal infection (105 PFU) of DENV2 D2S10, and animals were treated 24 hours post-infection with 20 µg of genetically-modified MAbs lacking effector functions, where the N297Q variant MAbs are fully aglycosylated and the LALA variant MAbs remain glycosylated but incapable of binding either FcγR or C1q P<0.01, and Table S1). In comparison, the EDIII-A-strand-specific MAb E76 N297Q showed partial protection (P<0.05), whereas MAbs E44 N297Q (EDII dimer interface) and E87 N297Q (EDIII C-C′ loop) provided no protection against lethal disease .Although secondary infection with a different DENV serotype is the greatest risk factor for severe DENV disease, DHF/DSS also has been reported following primary infection in vivo therapeutic potential. The neutralizing activity against DENV2 D2S10 of each of the MAbs was assessed using a flow cytometry-based assay with human monocytic U937 cells expressing DC-SIGN, a known attachment factor for DENV 50 in ng/ml of MAb). No significant difference was observed between each intact MAb and its modified variant. The NT50 of therapeutically effective MAbs E60 N297Q (EDII fusion loop) and 87.1 LALA (EDIII A strand) were 72 ng/mL and 24 ng/mL, respectively. In comparison, the NT50 of MAbs E44 N297Q (EDIII C-C′ loop) and E87 N297Q (EDII dimer interface), which bound other epitopes in EDII and EDIII and lacked therapeutic activity, were similar  .To determine why some MAbs had therapeutic activity in the virus-only lethal infection model but not in the context of antibody-enhanced infection, we examined several properties including epitope specificity, neutralization potency, and avidity. We first assessed whether neutralization potency correlated with ctively) Table 1.app) of binding, using a direct, virion-coated ELISA P<0.083), analogous to our neutralization data, we did not observe a relationship between MAb avidity and therapeutic efficacy  by MAbs targeting non-fusion loop epitopes , E18 N297Q (NT50 363 ng/mL) and E28 N297Q (NT50 544 ng/mL) , MAbs E18 N297Q and E28 N297Q failed to confer a therapeutic benefit following an antibody-enhanced lethal infection . MAb 82.11 LALA protected 50% (3/6) of animals following an antibody-enhanced infection, though this difference trended but did not attain statistical significance compared to non-treated control animals . In contrast to the experiments with non-fusion loop-specific MAbs, studies with MAbs targeting the same fusion loop epitope suggest that neutralization potency can predict therapeutic efficacy following an antibody-enhanced infection .As neutralization potency and avidity failed to correlate directly with the therapeutic efficacy of our modified MAbs across different epitopes, we investigated whether epitope specificity had greater predictive potential. As MAb E60 N297Q was highly protective even 48 hours following antibody-enhanced DENV infection 4 ng/mL) Table 1.in vitro neutralizing potency and in vivo therapeutic efficacy within fusion loop-specific MAbs, we generated a model of competitive displacement. Recent work has suggested that a significant fraction of the human anti-flavivirus E protein antibody response is directed against the fusion loop epitope in EDII 50 of 393 ng/mL) mouse MAb that binds to the fusion loop epitope P<0.01) when administered after a 4G2-enhanced infection, whereas MAb 82.11 LALA was less therapeutic (P<0.05), protecting 4 of 6 treated animals . None of the animals treated with MAb E18 N297Q succumbed to infection (P<0.05), although all demonstrated signs of illness . However, mice treated with MAb E28 N297Q all succumbed to 4G2-enhanced DENV2 D2S10 infection. These data support a model in which modified fusion loop-specific MAbs of sufficient avidity and neutralizing potency compete effectively for binding sites in the context of enhancing polyvalent DENV-immune serum or other fusion loop-specific MAbs to prevent disease.To explain the correlation between P = 0.64 by Friedman's analysis of data combined from seven experiments). In contrast, both MAb E60  and, surprisingly, MAb 87.1  altered binding of MAb 4G2; higher concentrations of MAb E60 and MAb 87.1 resulted in lower amounts of MAb 4G2 bound .We next evaluated directly whether MAb E60 (EDII fusion loop-specific) could effectively compete for binding with less potent fusion loop-specific MAbs, as compared to either therapeutic MAb 87.1 (EDIII A strand-specific) or non-therapeutic MAb E87 (EDII C-C′ loop-specific) that both target distinct epitopes. After directly coating DENV2 virions on microtiter plates, we added the moderately neutralizing mouse MAb 4G2 at 1 µg/mL mixed with increasing concentrations  of modified human MAbs followed by an anti-mouse, Fc-specific secondary MAb. Binding of mouse MAb 4G2 was not affected by the amount of bound E87  , 87.1 LALA  and E87 N297Q  in the following ratios: 100% 4G2; 95% 4G2 and 5% modified MAb; 85% 4G2 and 15% modified MAb; 75% 4G2 and 25% modified MAb. Each MAb combination was incubated with DENV2 D2S10 virus and used to infect K562 cells, a human erythroleukemic cell line that expresses FcγRIIA (CD32A) and is non-permissive in the absence of enhancing anti-DENV antibodies. Infection was monitored after 48 hours by intracellular DENV antigen staining and quantified by flow cytometry. Peak enhancement with MAb 4G2 occurred at 466 ng/mL and resulted in ∼7 to 15% of the cells becoming infected . However, when E60 N297Q or 87.1 LALA, MAbs that were effective therapeutically and lack the ability to engage Fcγ receptors, comprised only 5% of the MAb population, peak enhancement by 4G2 was reduced by 33% and 65%, respectively . Remarkably, when 4G2 and the modified MAbs were mixed in a 75%: 25% ratio, E60 N297Q and 87.1 LALA both reduced infection by 85% and 86%, respectively . In comparison, mixture of 5% of the non-therapeutic modified MAb E87 N297Q, reduced enhancement by only 21% , and by 57%  when a 75%: 25% mixture was used . Thus, highly avid, fusion-loop specific MAb E60 N297Q and A-strand-specific MAb 87.1 LALA minimized in vitro enhancement, presumably by preventing binding of the enhancing fusion loop-specific MAb 4G2. Furthermore, the ability of the modified MAbs to prevent 4G2-mediated enhancement in vitro correlated with in vivo therapeutic activity in the context of anti-DENV polyvalent serum-enhanced infection.We next tested whether modified MAbs targeting the same or different epitopes with respect to the enhancing MAb 4G2 (fusion loop-specific MAb) could suppress enhancement in vivo. We administered 20 µg of MAb 4G2 24 hours prior to infection with DENV2 D2S10 and then treated mice one day post-infection with 20 µg of MAb E60 N297Q (EDII fusion loop-specific)), 87.1 LALA (EDIII A strand-specific), or E87 N297Q (EDIII C-C′-loop-specific). While MAb E87 N297Q was not therapeutically protective against a 4G2-enhanced lethal infection, E60 N297Q and 87.1 LALA protected against mortality in 6 of 6 and 5 of 6 animals, respectively . Thus, potently neutralizing, fusion loop-specific (E60 N297Q) and A strand-specific (87.1 LALA) MAbs both prevent antibody-enhanced disease, likely by displacing binding of enhancing MAbs that target the fusion loop epitope.Given the results in K562 cells with mixtures of modified MAbs and decreased 4G2-mediated enhancement, we wanted to evaluate further this relationship in vitro. We selected the two most therapeutically effective, modified MAbs (E60 N297Q (EDII fusion loop) and 87.1 LALA (EDIII A strand)), and mixed them with the intact parent MAbs in the following proportions: 100% intact MAb, 90% intact and 10% modified MAb, 75% intact and 25% modified MAb, 50% of each MAb, 25% intact and 75% modified MAb, 10% intact and 90% modified MAb, and 100% modified MAb. While several mixtures of E60 parent:E60 N297Q showed reduced enhancement, only the combination of 10% intact:90% modified was non-enhancing in vitro, suggesting that the majority of the antibody mixture must not bind to Fcγ receptors in order to abolish enhancement of DENV infection when MAb pairs are of comparable neutralizing potency and avidity , the majority of the mixture must lack the capacity for binding FcγR to avoid enhancement. Thus, a low stoichiometric threshold of binding is likely sufficient for enhancement of infection and disease.Using combinations of intact E60 and modified E60 N297Q (EDII fusion loop-specific MAb), we evaluated whether the requirement for 90% of the MAb mixture to lack FcγR binding for suppression of enhancement in vitro relationship to predict the ability of modified MAbs to be therapeutically effective in vivo in the context of immune serum-enhanced DENV infection. Initially, using DENV1-immune mouse serum, we identified the serum dilution (1∶180) responsible for peak enhancement of DENV2 D2S10 in K562 cells  therapeutic activity in vivo were more efficient (P<0.05) at suppressing ADE in K562 cells than MAbs that were less therapeutically active , yet physiologically relevant concentrations (1 and 2 µg/mL) of modified MAb , while non-binding, DENV4-specific MAb 22.3 LALA did not .As the K562 cell-based assay with mouse polyclonal anti-DENV1 serum and modified MAbs appeared to predict in vivo, we administered normal human serum (NHS) or enhancing amounts of anti-DENV4 human immune serum 24 hours prior to a sub-lethal infection with DENV2 D2S10, and tested the therapeutic efficacy of the modified MAbs. As expected, all mice pre-treated with NHS survived infection without any signs of morbidity. All mice receiving enhancing anti-DENV4 human immune serum and treated with a modified MAb  survived lethal enhanced infection with minimal signs of disease , which itself is a function of epitope accessibility on the virion, mechanism of inhibition, and avidity of binding in vivo following DENV enhancement by polyvalent mouse serum. While the avidity of binding to solid-phase DENV2 for 82.11 LALA and E60 N297Q was comparable, E60 N297Q is ∼2.5 fold more neutralizing, suggesting that the two MAbs might bind overlapping yet slightly distinct epitopes, or that the ensemble of viral conformations in solution in vivo compared to MAb E76 N297Q. Although further study is warranted, our data suggest that within an epitope class, there is a direct relationship between MAb avidity and neutralization potential in vitro and therapeutic efficacy in vivo.Multiple parameters, including neutralization potency, avidity and epitope specificity, affect whether a modified MAb is therapeutic against an antibody-enhanced DENV infection. In our panel, in addition to binding to either the fusion loop or A-strand epitope, a therapeutic MAb needed to be strongly neutralizing  were completely protective following antibody-enhanced infection with DENV1-immune mouse serum. This observation suggests a direct interplay between the enhancing antibodies in polyvalent serum and the neutralizing therapeutic MAbs that determines outcome. Thus, a second parameter affecting therapeutic efficacy is the ability of a modified MAb to out-compete the enhancing antibodies in polyvalent immune serum for binding to the virion. This concept is supported by functional data in vitro ADE experiments using K562 cells and non-modified MAbs, we can conclude that all MAbs (excluding E44 as it was not available for these studies) have the capacity to neutralize infection via a post-attachment mechanism at saturating concentrations . Following uptake via FcγR, antibody-enhanced DENV may still be neutralized by MAbs that block post-attachment – this phenomenon of trans-dominant neutralization of ADE by MAbs was described previously with the anti-WNV MAb E16 Figure S2, right side of the curve), most likely by blocking fusion, a critical step required for release of the DENV genome into the cytosol following receptor-mediated endocytosis. To distinguish this post-attachment neutralization pattern from a MAb that blocks via a pre-attachment mechanism and cannot prevent infection in a K562 assay, we can compare these data to the effects of anti-fusion loop MAb E60 on WNV infection. MAb E60 cannot prevent WNV infection in K562 cells even at saturating concentrations . The inability of MAb E60 to neutralize WNV in K562 cells occurs because WNV virions are present in a mature state to a far greater degree than DENV and therefore have a lower stoichiometry of binding for the fusion loop epitope in vivo, it is unlikely that the modified MAbs circulate at saturating concentrations, given the large amount of DENV virus and antigen present. Thus, the relevant question becomes which MAbs can reduce enhancement  most efficiently when the modified MAbs are at sub-saturating concentrations. Under these conditions, therapeutically effective MAbs (87.1 LALA and E60 N297Q) show greater efficacy than the other MAbs evaluated, likely due to their ability to compete for binding with enhancing antibodies, which interferes with FcγR crosslinking and limits DENV uptake and infection.An additional parameter that likely affects therapeutic efficacy of a MAb against antibody-enhanced DENV infection is its mechanism of action: whether the MAb binds prior to or following attachment of the virion to the target cell. However, the interpretation is not straightforward, as mechanism of neutralization of a given MAb may be affected by several variables: (a) stoichiometry and relative fractional occupancy at a given concentration in vitro enhancement when E87 N297Q comprises 25% of the antibody mixture. Despite this, E87 N297Q did not have therapeutic activity in vivo when 4G2 was used as the enhancing MAb. In addition, MAb E87 N297Q was less efficient at reducing MAb 4G2-enhanced infection than the therapeutically effective MAbs E60 N297Q and 87.1 LALA at any of the three conditions tested . Although more study is warranted, we speculate that the MAbs which bind epitopes that do not displace 4G2 enhancing MAbs did not protect in vivo because they failed to reach a stoichiometry that was sufficient for neutralization or do not block a post-attachment step .Remarkably, MAb 87.1 LALA, which maps to the A strand of EDIII, also was effective against anti-fusion loop MAb 4G2-mediated lethal DENV infection. While not as potent as MAb E60, MAb 87.1 appeared to compete with fusion loop-specific MAb 4G2 in the solid-phase DENV2 ELISA and also suppressed 4G2-induced enhancement in K562 cells. One possible explanation is that the A-strand epitope on EDIII is located next to the EDII fusion loop on adjacent DENV E proteins within a dimer in vivo; (b) cross-reactive enhancing MAbs present in DENV-immune human serum bind distinct epitopes, which do not interfere with binding and neutralization by modified MAbs targeting the EDII fusion loop, EDII dimer interface, EDIII A strand, or EDIII C-C′ epitopes. In possible support of this, recent studies of the human antibody repertoire against DENV suggest that anti-prM antibodies are a major component of the cross-reactive response and promote enhancement in vivoPrevious studies have established that the epitope repertoire of anti-flavivirus neutralizing antibody in mouse and human serum is different. Mice were found to generate neutralizing antibody responses against epitopes in EDIII (∼30%) in utero, it remains uncertain if similar principles apply during natural secondary DENV infection.One limitation of this study is the passive transfer model used to develop lethal DENV disease; we tested limited concentrations of enhancing polyvalent immune serum to distinguish between therapeutic and non-therapeutic modified MAbs. In the future, a more detailed dose-response study with different enhancing sera or MAbs will be needed to determine the range of efficacy of modified MAbs in mediating protection. In addition, while the passive transfer model of enhancement and protection may be relevant for infant DHF/DSS where potentially enhancing antibodies are received passively in vivo therapeutic efficacy in both situations. While further study is needed, this assay could be used to screen additional modified MAbs for potential use as DENV therapeutics. Overall, given these promising results, we suggest that further exploration of the utility of modified MAbs as therapy for DENV infections is warranted.In summary, our results suggest a model in which neutralization, avidity, and epitope specificity contribute to the therapeutic efficacy of modified MAbs. Despite the differences between mouse and human polyvalent antibody repertoires, the suppression-of-enhancement assay in K562 cells accurately predicted This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at the University of California Berkeley (R252-1012B).Aedes albopictus C6/36 cells (American Type Culture Collection) and titered by plaque assay on baby hamster kidney cells  in vitro neutralization and enhancement assays and sub-lethal in vivo infections were performed with non-concentrated virus. DENV2 D2S10 virus was concentrated by ultra-centrifugation for use in the virion ELISA and by centrifugation using 100,000 MWCO Amicon filters (Millipore) for lethal, virus-only in vivo infections. U937-DC-SIGN  and K562 cells were used for flow cytometry-based in vitro neutralization All viruses were propagated in H and VL region was performed as described previously H-VL regions of the recombinant MAbs are as follows: E18_VL KC254882 E18_VH KC254883 E28_VL KC254884 E28_VH KC254885 E44_VL KC254886 E44_VH KC254887 E60_VL KC254888 E60_VH KC254889 E76_VL KC254890 E76_VH KC254891 E87_VL KC254892 E87_VH KC254893.Mouse MAbs E60, E18 and E28 were generated against WNV E protein, but are cross-reactive with DENV E protein H-VL regions of the recombinant human MAbs are as follows: DV82VH KC294013, DV82VL KC294014, DV87VH KC294015, DV87VL KC294016.MAbs 87.1 and 82.11 are fully human MAbs, and their generation has been described previously in vitro suppression-of-ADE assay were de-identified and pre-collected as part of the Nicaraguan Pediatric Dengue Hospital-based Study The single DENV1- and DENV3-immune human sera samples used in the in vitro suppression-of-ADE and in vivo ADE experiments was a gift from Dr. Aravinda de Silva (University of North Carolina (UNC), Chapel Hill) and were received as de-identified and pre-collected samples, and as such were not considered part of human subjects research by the IRB at UC Berkeley. Convalescent DENV-immune sera were obtained at UNC Chapel Hill from volunteers who had experienced natural DENV infections during travel abroad. The protocol for recruiting and collecting blood samples from returned travelers was approved by the IRB of UNC Chapel Hill. Written informed consent was obtained from all subjects before collecting blood.The primary DENV4-immune human serum sample used for All procedures were pre-approved and conducted according to UC Berkeley Animal Care and Use Committee guidelines. AG129 mice 5 PFU of DENV1 strain 448 (gift from S. Kliks). Six to eight weeks post-infection, mice were sacrificed, and whole blood was collected by terminal cardiac puncture. Serum was isolated from whole blood by centrifugation, heat inactivated, and stored at −80°C.AG129 mice were infected intraperitoneally (i.p.) with 10DENV2 D2S10 enhanced disease: AG129 mice were administered either 25 µl mouse anti-DENV1-immune serum or 200 µl human anti-DENV-4 immune serum or 20 µg of MAb 4G2  i.p. 24 hours prior to infection with an intravenous (i.v.) sub-lethal, 105 PFU dose of DENV2 D2S10. DENV2 D2S10 virus-only lethal disease: AG129 mice were infected i.v. with 4×106 PFU of DENV2 D2S10. Treatment model: Mice were administered 25 µl of DENV1-immune serum on Day −1, 105 PFU of DENV2 D2S10 on Day 0, and 20 µg of modified MAbs in a final volume of 100 µl i.v. 24 hours following infection (Day +1). All animals were monitored carefully for morbidity and mortality for 10 days following infection.50 titers were calculated as described previously 50 titer is the average of between 3 and 5 individual experiments, with the exception of E44 N297Q (1 experiment).The neutralization titer of each parent and modified MAb was measured using the U937-DC-SIGN flow cytometry-based neutralization assay as described Polyvalent serum: Enhancement curves were generated as described in MAb competition: Intact and modified MAbs were pre-mixed in different ratios at a starting concentration of 40 µg/mL. Eight 3-fold dilutions were incubated in either duplicate or triplicate with DENV2 D2S10 at an MOI of 0.1 in a 1∶1 ratio before being added to K562 cells for an enhancement of infection assay 4 K562 cells. The cells were washed 2 hours following infection and fixed and stained for viral antigen. Relative infection was expressed as the average percent infection for each duplicate divided by the percent infection measured without modified antibody .DENV-immune serum was diluted to the concentration that resulted in the greatest enhancement of DENV2 infection in K562 cells. DENV2 D2S10 virus at an MOI of 0.1 and serum were mixed together in equal volumes for 30 to 45 minutes at 37°C. Modified MAbs were prepared in five 2-fold dilutions beginning at 2,000 ng/mL, added to the polyvalent serum/virus mixture, and incubated for an additional 30–45 minutes prior to the addition of 5×10Avidity ELISA: DENV2 D2S10 virus was isolated by ultra-centrifugation at 53,000× g for 2 hours at 4°C and resuspended in cold PBS with 20% FBS. Concentrated virus was diluted to 5×104 pfu in carbonate coating buffer, pH 9.6, and 50 µl was added to each well of a 96-well flat-bottomed plate as described previously Competition ELISA: Ninety-six-well flat-bottomed plates were coated with DENV2 as described above. Mouse MAb 4G2 was diluted to 1 µg/mL and mixed with human mAb diluted to 10, 1 and 0.1 µg/mL in a separate 96-well plate, and 100 µl of the mixture was added. After one hour, the plates were washed and incubated with goat anti-mouse Fcγ-specific biotinylated secondary (Jackson) followed by Streptavidin-AP (Invitrogen) and the plates were developed with PNPP as described above.50 titers of non-modified and modified MAb pairs was conducted using a Wilcoxon rank-sum analysis. Comparison of survival rates was conducted using a non-parametric log rank test. A Spearman rho (ρ) was calculated to assess correlations between modified MAb NT50 titer, avidity, and therapeutic efficacy . A Kruskal-Wallis test was used to compare 4G2 binding across increasing concentrations of human MAb, and a Friedman's analysis  was conducted by combining all data for each MAb tested. A Wilcoxon rank-sum test was used to compare differences in the percent reduction of 4G2-enhanced D2S10 infection in K562 cells with different mixtures of modified MAb as well as in the enhancement-suppression assay using mouse DENV1-immune serum to compare relative infection between therapeutic MAbs and non-therapeutic MAbs. A sign rank test was used to determine whether 1,000 ng/mL of modified MAb could reduce an infection enhanced with DENV4-immune serum significantly lower than 50%.All graphs were produced using GraphPad Prism 5 software . Statistical analysis was performed using Stata v10  and Prism 5 software. Comparison between NTFigure S1Neutralizing potency contributes to competitive binding of fusion-loop specific MAbs.A.Mouse MAb 4G2 (fusion loop-specific) was incubated at 1 µg/mL with anti-fusion loop MAbs 82.11, E18 or E28 at 10, 1 or 0.1 µg/mL human MAb prior to addition to DENV2-virion coated plates . Anti-mouse, Fc-specific secondary MAb was then added, followed by PNPP substrate. Optical density (OD) values are shown on the y-axis and were calculated after subtracting the average background (binding of mouse Fcγ-chain-specific secondary antibody). Statistically significant differences in 4G2 binding across the different human MAb concentrations were calculated using a Kruskal-Wallis test from triplicate values within each experiment. These data are representative of three independent experiments. B–D. MAb 4G2 was pre-mixed with MAb E40 N297Q, MAb E18 N297Q or MAb E28 N297Q in ratios of 95% 4G2/5% modified MAb (B), 85% 4G2/15% modified MAb (C), or 75% 4G2/25% modified MAb (D). For each 4G2/modified MAb mixture, a Gaussian distribution was used to fit each enhancement curve. The area under the curve (AUC) was calculated for each curve, and relative infection was expressed by dividing the AUC in the presence of modified MAbs by the AUC measured with 4G2 only (no modified MAb). The data displayed are the average of three to seven independent experiments +/− SEM, and comparison between the MAb combinations E60 N297Q/4G2 and E18 N297Q/4G2 or E28 N297Q/4G2 was performed using a Kruskal-Wallis test.(TIF)Click here for additional data file.Figure S2In vitro ADE assay with fusion loop-specific MAb 4G2 and modified MAbs. MAbs E60 N297Q (A), 87.1 LALA (B) and E87 N297Q (C) were mixed with MAb 4G2 at the concentrations specified in a starting dilution of 40 µg/mL. The percent infection of K562 cells is shown on the y-axis and log antibody concentration (ng/mL) on the x-axis. A Gaussian distribution was used to fit each curve separately.(EPS)Click here for additional data file.Figure S3Modified MAbs reduce in vitro enhancement by human DENV-immune serum. DENV1-immune (A), DENV3-immune (B), and DENV4-immune (C) human serum were diluted 1∶540 (peak enhancement identified in (EPS)Click here for additional data file.Table S1Therapeutic efficacy of modified MAb variants targeting different epitopes.(DOC)Click here for additional data file.Table S2Therapeutic efficacy of modified MAb variants targeting the fusion loop.(DOC)Click here for additional data file.Table S3Therapeutic efficacy of modified MAb variants following 4G2-enhanced, lethal DENV2 D2S10 infection.(DOC)Click here for additional data file.


KCNQ4 gene lead to DFNA2, a subtype of autosomal dominant non-syndromic deafness that is characterized by progressive sensorineural hearing loss across all frequencies. Despite recent advances in the identification of pathogenic KCNQ4 mutations, the molecular aetiology of DFNA2 remains unknown. We report here that decreased cell surface expression and impaired conductance of the KCNQ4 channel are two mechanisms underlying hearing loss in DFNA2. In HEK293T cells, a dramatic decrease in cell surface expression was detected by immunofluorescent microscopy and confirmed by Western blot for the pathogenic KCNQ4 mutants L274H, W276S, L281S, G285C, G285S, G296S and G321S, while their overall cellular levels remained normal. In addition, none of these mutations affected tetrameric assembly of KCNQ4 channels. Consistent with these results, all mutants showed strong dominant-negative effects on the wild-type (WT) channel function. Most importantly, overexpression of HSP90β, a key component of the molecular chaperone network that controls the KCNQ4 biogenesis, significantly increased cell surface expression of the KCNQ4 mutants L281S, G296S and G321S. KCNQ4 surface expression was restored or considerably improved in HEK293T cells mimicking the heterozygous condition of these mutations in DFNA2 patients. Finally, our electrophysiological studies demonstrated that these mutations directly compromise the conductance of the KCNQ4 channel, since no significant change in KCNQ4 current was observed after KCNQ4 surface expression was restored or improved.KCNQ4, a voltage-gated potassium channel, plays an important role in maintaining cochlear ion homoeostasis and regulating hair cell membrane potential, both essential for normal auditory function. Mutations in the  V7) plays important roles in brain, heart, kidney and the inner ear. Mutations in four of the five KCNQ channels cause inherited human diseases, including cardiac arrhythmia, epilepsy, deafness, etc. V7.4) is predominantly detected in the inner ear and the central auditory pathways IK,n+ recycling in the cochlea + efflux pathway from outer hair cells KCNQ4 cause DFNA2, a subtype of autosomal dominant non-syndromic deafness that is characterized by progressive sensorineural hearing loss The KCNQ family of voltage-gated potassium channels KV plays imKCNQ4 mutations have been identified in DFNA2 patients (DFNA2 mutations) Xenopus laevis oocytes and various cell lines have shown that these mutations lead to loss of KCNQ4 currents et al. also found that five other DFNA2 mutants, L274H, W276S, L281S, G285C and G321S, are trafficking deficient, although no experiment was conducted to determine whether or not the trafficking phenotype was dominant Over the last two decades, various pathogenic +-filter (GYG); G296S is located in a group of five highly conserved amino acids connecting the pore-loop and the transmembrane domain S6; G321S is at the junction of the S6 domain and the C-terminal of the KCNQ4 channel. Given the structural and functional significance of the amino acids affected, it is not surprising that these mutations lead to loss of KCNQ4 function. However, the finding that loss of KCNQ4 currents may be caused by decreased cell surface expression is striking and opens a series of important questions in the molecular aetiology of DFNA2. What are the underlying mechanisms for the loss of KCNQ4 currents in DFNA2? Is it because of decreased cell surface expression or impaired conductance of KCNQ4 channels or both? Is decreased surface expression a consequence of degradation or intracellular retention of DFNA2 mutants? Is it possible that DFNA2 mutants are intracellularly retained otherwise functional? Could surface expression of DFNA2 mutants be restored? Most importantly, could restoration of KCNQ4 surface expression rescue the function of these mutant channels? The answers to these questions are fundamental to our understanding of the molecular mechanisms underlying hearing loss in DFNA2 and will lay important ground work for a rational design of therapeutic treatments.Like other voltage-gated potassium channel, KCNQ4 channels consist of six transmembrane domain, a pore-forming region, and two intracellular termini. Most of the DFNA2 mutations are clustered around the pore region of the KCNQ4 channel etc. ATP) Generation of KCNQ4 channels on the cell surface requires proper folding, assembly and trafficking of KCNQ subunits. Despite the functional significance of the KCNQ4 channel, little is known about the molecular mechanisms that control these processes. We have recently demonstrated that the cellular level of the KCNQ4 channel is regulated by the HSP90 chaperone pathway In this study, we investigated the effects of seven loss-of-function DFNA2 mutations, L274H, W276S, L281S, G285C, G285S, G296S and G321S, on the overall cellular level and on the cell surface expression of the KCNQ4 channel using both immunofluorescent and quantitative biochemical approaches. We tested whether these mutations affect subunit interaction and whether they have dominant-negative effects on the WT KCNQ4 function. We also explored the potential of HSP90 overexpression in rescuing cell surface expression of DFNA2 mutants L281S, G296S and G321S. Finally, we tested whether the function of DFNA2 mutations can be rescued by restoration of KCNQ4 surface expression in HEK293T cells mimicking the heterozygous KCNQ4 condition of DFNA2 patients.All chemicals were from Sigma-Aldrich ; media and reagents for cell culture were from Invitrogen , unless otherwise indicated.KCNQ4 (NM_004700) was cloned in pCMV6-XL5 vector and then tagged with a Myc or a modified HA epitope in the first extracellular loop of the KCNQ4 channel as described previously Primary antibodies used in this study were anti-HA , anti-Myc , anti-GAPDH , anti-HSP90β . The secondary antibodies, including the anti-goat-horseradish peroxidase , antimouse-HRP (715-035-151) and antimouse IgG-FITC (115-095-146) were from Jackson ImmunoResearch Laboratories Inc. .HEK293T cells (Sigma-Aldrich) were used for all experiments. These cells were maintained according to the manufacturers' instruction. All transfection were carried out using Lipofectamine 2000 as described by the manufacturer (Invitrogen). Following transfection, the cells were incubated at 37°C for 24 hrs.HEK293T cells were cultured on glass cover slips and transfected with KCNQ4 channels in pIRES2-DsRed2 (0.4 μg per well in 6-well plates). Twenty-four hours after transfection, the cells were fixed in 4% paraformaldehyde for 5 min., followed by three washes in PBS, and blocked in StartingBlock blocking buffer  for 10 min. For permeabilization, the blocking buffer was supplemented with 0.1% Triton X-100. The cells were then treated with mouse monoclonal anti-HA antibody (1:500 dilution) for 1 hr and washed three times with PBS before incubation with goat-antimouse IgG-FITC . After a brief wash with PBST (PBS plus 0.05% Tween 20), the cells were incubated in the dark with DAPI for 5 min., followed by three washes with PBST, and then mounted in ProLong Gold Antifade Reagent (Invitrogen) on glass slides. Fluorescent images were captured using Carl Zeiss LSM510 confocal microscope. All procedures were performed at room temperature.g for 10 min. at 4°C and incubated with primary antibodies as indicated at 4°C for 16 hrs. The protein complexes were isolated and purified using Dynabeads Protein G  following manufacturer's protocol and analysed by Western blot (see below).Transfected cells were lysed in NP40 lysis buffer supplemented with protease inhibitor cocktail , on ice for 30 min. Cell lysates were then cleared by centrifugation at 18,400 × in situ in 6-well culture plates and treated with mouse monoclonal anti-HA or anti-Myc antibodies (1:500 dilution) for 30 min. to label KCNQ4 channels on the cell surface. Following three washes with PBS, the cells were lysed with NP40 lysis buffer supplemented with protease inhibitor cocktail , on ice for 30 min. The cell lysate was transferred to fresh tubes and centrifuged at 14,000 rpm for 10 min. at 4°C. Surface KCNQ4 proteins were captured using Dynabeads Protein G as instructed by the manufacturer. Proteins eluted from Dynabeads were analysed by Western blot (see below).Twenty-four hours after transfection, cells were washed twice with PBS Proteins were separated on Criterion™ TGX Precast Gels  and transferred to a nitrocellulose membrane (Bio-Rad Life Science). The membrane was probed with appropriate primary antibodies followed by incubation with HRP-conjugated secondary antibodies and visualized by SuperSignal West Pico Chemiluminescent Substrate . Chemiluminescent signals were collected by ChemiDoc XRP Imaging System and analysed by Quantity One software (Bio-Rad Life Science). Each band was quantified as the total pixel value after subtraction of the background and normalized to the loading control protein GAPDH.2, 1 mM MgCl2, 10 mM HEPES, pH7.4, and osmolarity of 303 mmol/kg). Only healthy looking attached cells expressing DsRed fluorescent marker were used for recordings. Glass electrodes with resistance ranging from 1.5 to 3.0 MΩ and filled with internal solution  were used. Data acquisition was performed using a HEKA EPC-10 amplifier and HEKA PatchMaster software  with high band Bessel filter set to 10 kHz and low band filter set to 0.2 kHz. The protocol consisted of holding for 1 sec. at −80 mV, followed by depolarization from −80 to +50 mV in 10 mV steps for 1.5 sec., −50 mV for 1 sec. followed by 1 sec. at −80 mV, with 5 sec. in between each testing sequence. Whole-cell current densities (pA/pF) were calculated as the maximal current (pA) divided by the cell capacitance, C-slow (pF). Each day at least five cells expressing WT KCNQ4 were recorded as internal control group. All recordings were performed at room temperature.HEK293T cells were trypsinized 24 hrs after transfection, seeded onto poly-L-lysine-coated glass coverslips, and maintained under normal growth condition for about 4 hrs. Before recording, cells were extensively washed with external solution . Significance was reported as *P ≤ 0.05 and **P ≤ 0.01. Whole-cell current data were exported from PatchMaster and current traces were plotted using Origin v8.6 software .Biochemical results were presented as mean ± SD of at least three independent experiments. The measurements were statistically analysed using two-tailed unpaired Student's KCNQ4 gene. Both the WT and mutant KCNQ4 channels were tagged with a HA epitope at the extracellular loop between the S1 and S2 domains and expressed in HEK293T cells. Immunofluorescent analyses were carried out using a primary antibody specific to the HA tag and a FITC-conjugated secondary antibody in both permeabilized and non-permeabilized cells. Confocal microscopy demonstrated strong green fluorescence with similar cellular distribution along the secretory pathway in all permeabilized cells expressing the WT or a mutant channel , 26.48 ± 6.03% (W276S), 7.16 ± 1.93% (L281S), 35.77 ± 6.08% (G285C), 18.47 ± 5.30% (G285S), 1.78 ± 1.11% (G296S) and 31.51 ± 5.72% (G321S) C and D. KCNQ4 alleles, encoding equal amount of the WT and mutant KCNQ4 subunits. To examine whether DFNA2 mutations affect heteromeric assembly between the WT and mutant subunits, mutant HA-KCNQ4 channels were co-expressed individually with the WT Myc-KCNQ4 in HEK293T cells at a ratio of 1:1 to mimic the heterozygous condition of DFNA2 patients. The cell lysates were analysed by reciprocal co-immunoprecipitation assays. In forward experiments, interactions between the WT and mutant subunits were assessed by the amount of the WT Myc-KCNQ4 subunit co-precipitated with mutant HA-KCNQ4 subunits  a DFNA2 mutant alone; (ii) a DFNA2 mutant plus HSP90β; (iii) a mutant and the WT KCNQ4 at a ratio of 1 to 1 to mimic the heterozygous condition of the DFNA2 patients; (iv) a mutant and the WT KCNQ4 at ratio of 1:1 plus HSP90β. Whole-cell currents were also recorded from cells expressing the WT KCNQ4 under the same conditions as control groups. Outward currents recorded from cells expressing the WT KCNQ4 alone were similar to whole-cell currents reported previously 45. Compared with these currents (n = 30) for the WT channel, but 15.51 ± 2.01 pA/pF (n = 15) for the mutant L281S, 11.82 ± 2.7 pA/pF (n = 15) for G296S, 14.89 ± 1.77 pA/pF (n = 12) for G321S, and 6.31 ± 1. 01 pA/pF (n = 5) for non-transfected cells (n = 11) for WT/L281S, 17.58 ± 2.7 pA/pF (n = 12) for WT/G296S and 17.28 ± 2.64 pA/pF (n = 7) for WT/G321S (n = 30). Our data demonstrated that the function of DFNA2 mutants could not rescue by co-expression of the WT KCNQ4 subunit; instead, the mutants had dominant-negative effects on the WT KCNQ4 currents , although the difference did not achieve statistical significance , but it is still much smaller than the WT level  and translocate to the plasma membrane. In human cells, these processes are controlled by a sophisticated molecular network and monitored by the protein quality control system KCNQ4 gene have been identified in DFNA2 families. A genotype–phenotype correlation has been observed in which missense mutations are associated with a younger onset and severe to profound hearing loss across all frequencies; while deletions are associated with a later onset and milder hearing loss that mainly affects high frequencies Various missense mutations and deletions in the KCNQ4 result in progressive hearing loss that is paralleled by a selective degeneration of the hair cells and spiral ganglion neurons KCNQ4 to age-related hearing loss, which may be attributable to the elevated expression of a causative KCNQ4 splice variant during ageing etc. Sensorineural hearing loss in DFNA2 patients progresses over years +-filter in the KCNQ4 channel. Thus, it was uncertain whether these intracellularly retained KCNQ4 mutants could assemble channels with normal K+ conductance. To answer this question, whole-cell currents were recorded from cells in which the KCNQ4 surface expression had been restored by overexpressing HSP90β. We found that the conductance of the KCNQ4 channel was compromised by these three mutations, as no significant improvement in KCNQ4 current density was observed in these cells. Therefore, our data confirmed that decreased cell surface expression and impaired conductance of the KCNQ4 channel are two independent mechanisms underlying hearing loss in DFNA2; that restoration of KCNQ4 surface expression by overexpression of HSP90 was not sufficient to rescue the channel function in HEK293T cells mimicking the heterozygous condition of DFNA2 patients. These findings together laid important frame work for future studies towards functional rescue of pathogenic KCNQ4 mutations.Another important but unsolved issue in the molecular aetiology of DFNA2 was whether DFNA2 mutations disrupt the conductance of the KCNQ4 channel, in addition to their effects on KCNQ4 surface expression. Unlike the mutations G285C and G285S, mutations L281S, G296S and G321S do not result in alterations in the signature sequence of the K


Vitamin D and the components of humoral immunity play important roles in human health. Older people have lower 25-hydroxyvitamin D (25(OH)D) serum levels than younger adults. We aimed to determine the levels of 25(OH)D serum concentrations in healthy senior citizens and to study their relationship to the levels of components of humoral immunity.A total of 1,470 healthy Swiss men and women, 60 years or older, were recruited for this study. A total of 179 subjects dropped out of the study because of elevated serum concentrations of C-reactive protein. Fasting blood sera were analyzed for 25(OH)D with the high-performance liquid chromatography (HPLC) and for parathyroid hormone (PTH), immunoglobulins and complement C4 and C3 concentrations with immunoassays. The percentage of participants in each of the four 25(OH)D deficiency groups - severely deficient (<10 ng/ml), deficient (10 to 20), insufficient (21 to 29 ng/ml) and normal (>=30 ng/ml) - were statistically compared. The relationship of the major components of the humoral system and age with 25(OH)D levels was also assessed.P = 0.01) and with C4 (P = 0.02), yet were inversely related to levels of IgG1 and IgA (P < 0.05) and C3 (P = 0.01). Serum levels of total IgA, IgG, IgG2 and IgG4 peaked together with 25(OH)D during late summer.About 66% of the subjects had insufficient levels of 25(OH)D. Normal levels of 25(OH)D were found in 26.1% of the subjects of which 21% were males and 30.5% were females . Severely deficient levels of 25(OH)D were found in 7.98% of the total study population. Low levels of 25(OH)D were positively associated with IgG2 (Approximately two-thirds of the healthy, older Swiss population presented with Vitamin D insufficiency. The incremental shift in IgA and C3 levels might not necessarily reflect a deranged humoral immune defense; however, given the high prevalence of vitamin D deficiency, the importance of this condition in humoral immunity will be worth looking at more closely. This study supports the role of vitamin D in the competent immune system. The biological role of fat-soluble vitamin D has been shown in recent studies to extend far beyond its role in calcium homeostasis and bone health -4. Curre2D3) blocks the induction of T-helper-1 (TH1)-cell cytokines  and promotes TH2-cell responses. This activity partly explains the decreased B-cell proliferation, plasma-cell differentiation and IgG secretion [2D3 on B cell function have been researched for many years [Experimental, epidemiological and clinical studies have shown inverse associations of low vitamin D status with longevity and with retarding immunosenescence . 1,25-diecretion ,9,10, whny years ,11-14; fny years . Althoug2D). Humans acquire much of their vitamin D through a photosynthetic reaction in the skin with a small amount via digestive intake [Throughout the life span, a significant proportion of humans have insufficient (<30 ng/ml) or deficient <20 ng/ml) serum 25(OH)D levels. 25(OH)D severe deficiency may be defined as serum concentrations <10 ng/ml ,17, defi0 ng/ml se intake . The acqe intake .The European population is known for transient reductions of 10 to 20% of individual 25(OH)D serum levels measured during the winter months compared to the summer months, an observation that is particularly prominent in the older people .The present study was initiated (i) to study the prevalence of insufficiency and deficiency levels in healthy older people and (ii) to assess the impact of these deficiencies on immunoglobulin and complement components C4 and C3 production.http://www.seniorlabor.ch). The study participants were contacted through newspaper advertisements and different clubs and associations that had a high probability of having healthy, older members  and through the personal contacts of collaborators of the study organization.Consecutive subjectively healthy, older volunteers aged 60 years and older were recruited between February 2009 and July 2010 as part of the Senior Labor Study, which is an ongoing investigation in the canton of Berne (Switzerland) initially aimed at creating appropriate reference intervals (RIs) for several analytes in older persons . Of the 1,470 participants, 179 with a high total leukocyte count (>11.5 G/L) and/or high C-reactive protein levels (>5 mg/L) were excluded from further analysis since such laboratory cutoffs are strong indicators of the presence of systemic inflammation, mostly for upper respiratory tract infections. Of the remaining 1,291 subjects, 558 were between 60 and 69 years old, 498 between 70 and 79 years old and 235 were 80 years old or older , Bern, Switzerland.2 and 25(OH)D3  were distinguishable using this method; for this study, only the latter, more stable animal-origin 25(OH)D3 levels are reported. The US National Institute of Standards and Technology (NIST) calibration standard reference material (http://www.nist.gov) was consistently followed with each test batch, and only the values complying with the requirements were released; the inter- and intra-assay variations were <5%. The baseline total 25(OH)D levels were categorized as normal, insufficient, deficient and severely deficient using levels adapted from the definitions proposed earlier [All assays were done at our institution. For 25(OH)D concentration measurements, we used the LaChrom Elite apparatus provided by Hitachi . 25(OH)D earlier ,24. The The levels of immunoglobulins, where applicable, were measured using the BN Prospec® Nephelometry System , incorporating stabilized human serum as the N Protein Standard SL. Subclass-specific sheep anti-human IgG1 and IgG2 were used in conventional nephelometry and anti-human IgG3 and IgG4 in this system is loaded on polystyrol particles to enhance sensitivity for quantitation of the cognate subclass. For C3 und C4, we used sodium azide preserved anti-C3c antibodies <6.4 mg/ml and anti-C4 <5.4 mg/ml. The company recommends application of the following reference intervals established with serum and/or plasma samples from healthy adults: C3/C3c of 0.9 to 1.8 mg/ml and C4/C4c of 0.1-0.4 mg/ml.P <0.05. The Kruskal-Wallis test was used to test for statistically significant associations of 25(OH)D and immunoglobulins in more than two groups. Further, the Mann–Whitney rank sum was used to statistically compare the levels of various immunoglobulins and complement components by categories of 25(OH)D levels . Univariate linear regression analysis was used to examine the relationship between 25(OH)D as the dependent variable and age, immunoglobulin and complement components as the independent variables.The serum concentration values of 25(OH)D, immunoglobulin classes, IgG subclasses and complements were statistically analyzed using SPSS version 17.0 for Microsoft Windows . All statistical analyses were based on two-sided hypothesis tests with a significance level of P <0.001) (Table P <0.001) D levels D levels and major humoral components of innate and acquired immunity. While levels of IgA inversely correlated to 25(OH)D, IgM nor of total IgG or IgE differed from changing 25(OH)D levels D severely deficient and the normal group.The The mean serum level of IgG2 significantly differed among 25(OH)D level groups Table A, B, wheP = 0.001), IgG2 (P = 0.003), IgG4 (P = 0.04), complement component C4 (P = 0.01), and complement component C3 (P = 0.006). Total IgG, IgG2 and IgG4 peaked together with 25(OH)D during August.Seasonal variation in immunoglobulin levels and complement components revealed differences according to the month samples were taken D status has been connected to humoral immunity features in healthy older subjects not receiving any real-time medical care; in fact, most studies reporting insufficient/deficient 25(OH)D levels have been performed in the context of disease, such as patients assigned to primary care , prostatHere, a majority (over 50%) of the older, subjectively healthy Swiss population presented with 25(OH)D insufficiency/deficiency. We observed a progressive decline in 25(OH)D levels with aging both in women and men, most likely due to lower sunlight exposure and a decline in balanced food composition for those not (yet) part of homes for retired, older persons, as those under study here. In the Osteoporotic Fractures in Men Study (MrOS) from North America, 31% of men aged 80 to 84 years had levels <20 mg/ml , and theviz. no risk considerations [In conjunction with these new insights, 25(OH)D testing requests received by medical analytical laboratories are surging worldwide . The preerations .Depending on latitude and climate, seasonal/monthly variations in serum levels of 25(OH) do occur  or remaiP-values under non-parametric conditions highlight the findings that incremental insufficiency progresses to deficiency with increasing age, a finding not taken into account by the Endocrine Society’s Clinical Guidelines [To establish reference intervals (RIs), different statistical procedures are commonly applied at each step. The sample size in the >80-year group of the present study exceeds the 100-observation barrier. Indeed, the significant idelines .The ability of vitamin D to influence normal human immunity is highly dependent on the 25(OH)D status of individuals, and may lead to an aberrant response to infection or autoimmunity in those with insufficient vitamin D. This article describes some of the recent developments with respect to vitamin D and the immune system, and possible clinical implications. The influence that 1,25(OH) vitamin D exerts on the immune system sparks interest for its potential for reducing infections and preventing allograft rejection after transplantation .2D3 in modulating immune responses [2D3in situ[3 to 1,25D3 in the vitamin D-dependent antimicrobial pathway [Beyond the risk for rickets and osteoporosis, other diseases are increasingly being noticed for their association with Vitamin D status . Therefoesponses ,45,46. SD3in situ. Recent D3in situ. A potenD3in situ,49, whic pathway .In vitro, the effects of 1,25(OH)2D3 on lymphocyte proliferation and cytokine production by PBMCs are pleiotropic [iotropic . Total siotropic , althougiotropic  nor in oThe majority (approximately two-thirds) of healthy older Swiss subjects present with vitamin D insufficiency. Showing the impact on serum concentrations of IgG1, IgG2, IgA and complement components C4 and C3, more so in severe cases of vitamin D deficiency, but also with insufficiency of 25(OH)D, this study supports a role of vitamin D in the immune system under immunocompetent conditions.2D, 1,25-dihydroxyvitamin D; 1,25(OH)2D3): 1,25-dihydroxyvitamin D3; 25(OH)D: 25-hydroxyvitamin D; GLP: Good laboratory practice; HPLC: High-performance liquid chromatography; IFNγ: Interferon gamma; MrOS: Osteoporotic Fractures in Men Study; NIST: National Institute of Standards and Technology; PTH: Parathyroid hormone; RIs: Reference intervals; RXR: Retinoid X receptor; TH1: T-helper-1; VDRs: Vitamin D receptors.1: 25(OH)The authors declare that they have no competing interests.UN, LR and MR participated in the design of the study. BS, CN, ZS, UN and LR drafted the manuscript and analyzed the data. BS, PM and CN performed the statistical analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/11/176/prepubMonthly variation of 25(OH)D and components of the humoral immune system in older Swiss. Monthly follow-up of mean ± 1 SD serum levels of 25(OH)D and humoral immune components. The IgE increment in summer likely reflects pollen exposure of the subjects. Number of samples collected per month are listed in brackets on the abscissa.Click here for filePercentage increase/decrease in level of 25(OH)D and humoral immune components in older Swiss. The percentage increase or decrease in seasonal variation of the humoral immune components showing significant correlation in seasonal variation with 25(OH)D, compared to the month of January (100%) is further illustrated here.Click here for file