PSMD9) single nucleotide polymorphisms (SNPs) rs74421874 [intervening sequence (IVS) 3+nt460-G>A], rs3825172 (IVS3+nt437-C>T) and rs14259 (E197G-A>G) are linked to: T2D, depression, anxiety, maturity-onset-diabetes-of the young 3/MODY3, obesity, waist circumference, hypertension, hypercholesterolemia, T2D-macrovascular disease, T2D-microvascular disease, T2D-neuropathy, T2D-carpal-tunnel syndrome, T2D-nephropathy, T2D-retinopathy and non-diabetic retinopathy. PSMD9 SNP rs1043307/rs14259 (E197G-A>G) plays a role in anti-depressant therapy response, depression and schizophrenia. We aimed at determining PSMD9 rs74421874/rs3825172/rs14259 SNPs potential linkage to primary insomnia and sleep hours in T2D families. We recruited 200 Italian T2D families phenotyping them for primary insomnia and sleep hours per night. PSMD9-T2D-risk SNPs rs74421874/rs3825172 and rs1043307/rs14259 were tested for linkage with insomnia and sleep hours. Non-parametric-linkage analysis, linkage-disequilibrium-model analysis, single-SNP analysis, cluster-based-parametric analysis, quantitative-trait and variant-component analysis were performed using Merlin software. To validate data, 1000 replicates were executed for the significant non-parametric data. PSMD9 rs74421874 (IVS3+nt460-G>A), rs3825172 (IVS3+nt437-C>T) and rs1043307/rs14259 (E197G-A>G) SNPs are linked to insomnia in our Italian families.Insomnia increases type-2 diabetes (T2D) risk. The 12q24 locus is linked to T2D, depression, bipolar disorder and anxiety. At the 12q24 locus, the Proteasome-Modulator 9 ( Insomnia is associated with increased risk for type-2 diabetes (T2D)6789PSMD9), contributing to T2D through rare variants [rs149556654 SNP (N166S A>G) and S143G A>G variant]PSMD9 is significantly linked via the common single nucleotide polymorphisms (SNPs) rs74421874 (IVS3+nt460 G>A), rs3825172 (IVS3+nt437 C>T) and rs14259 (E197G A>G) to T2D151726PSMD9 SNP rs1043307/rs14259 (E197G A>G) is implicated in anti-depressant therapy responsePSMD9 SNPs rs74421874 (IVS3+nt460 G>A), rs3825172 (IVS3+nt437 C>T) and rs14259 (E197G A>G) are linked to depressionPSMD9 is strongly implicated in depressionPSMD9 may well play a role in insomnia.Within the 12q24 locus654 SNP N6S A>G anrs14259 E7G A>G to IVS3+nt4 C>T and http://www.genecards.org/cgi-bin/carddisp.pl?gene=PSMD9&search=psmd9). Therefore, PSMD9 may have a pathogenic role in inflammation and autoimmunityPSMD9 variants impairing PSMD9 protein sequence and/or protein dosage may alter downstream effects of its functions as well as interaction with key receptors such as the IL6 receptor. As PSMD9 is part of a complex network of transcription and co-activators, reduced or increased PSMD9 gene may lead to different phenotypes, or to an underlying pathological factor such as inflammation contributing to the various phenotypes, and commonly shared by them. Its proven interaction with the IL6 receptor may potentially explain its pleiotropic role in multiple disorders, whose pathogenesis is mediated by inflammation. However, other possible explanations for a shared pathogenesis among neuro-psychiatric disorders exist. For example, adult neurogenesis might be a more plausible pathogenic mechanism for neuro-psychiatric disorders. The proteasome-mediated proteolysis plays a recognized role in neuronal development, synaptic plasticity, and protein quality control. A proteomic study has reported the positive finding of PSMD9 at both the level of neuronal cytosol as well as synapsisPSMD9 gene role in the ubiquitin-proteasome system and its proven protein interaction with dysbindin and dysbindin-related role in neuroplasticity is intriguingPSMD9 is ubiquitously expressed and highly concentrated in eukaryotic cells as part of the 26S proteasome complex, which degrades intracellular proteins into antigenic peptides in the immune response and antigen presentation by MHC class I cells (PSMD9-depression-risk SNPs rs74421874 (IVS3+nt460 G>A), rs3825172 (IVS3+nt437 C>T) and rs14259 (E197G A>G) for linkage with insomnia and sleep hours in the 200 Italian T2D families.In light of these data, our object in the present study was to test the The subjects were all recruited from central Italy following the Helsinki-declaration guidelines. Subjects gave written informed consent. The Penn State Institutional Review Board approved the study.The Italian T2D families with affected sib-pairs were recruited via standardized interview from Rome. They are genetically homogeneous, and all have been Italian for at least three generations; T2D families have a T2D-family history. We excluded families with identical twins and siblings with uncertain identical paternity. The Italian family dataset has provided useful genetic information in T2D and associated phenotypes124445464748495051525354555657585960The Italian T2D families with mainly affected sib-pairs were recruited via standardized interview. The 200 Italian families are mainly constituted by 360 ungenotyped parents of 180 affected sib-pairs  for a total of 380 affected siblings, plus 20 families inclusive of 101 genotyped family members with an average family size of 5.05. The total of genotyped family members is 481, including 245 female subjects and 236 male subjects. The median age was 63. T2D was diagnosed based on the fasting criteria of blood glucose ≥126 mg/dl on at least two occasions, or a random glucose of ≥200 mg/dl associated with symptoms of diabetes, exclusion of type 1 diabetes, and a positive family history for non-insulin-dependent diabetes/T2D. T2D was present in 94.10% of the families’ members. Major depression and generalized anxiety disorder were diagnosed based on the subject meeting the DSM-IV criteria during his/her lifetime. Generalized anxiety disorder was present in 52.50% of families and major depression in 29.90% of families.We characterized the Italian families for the presence or absence of insomnia based on the DSM-IV diagnosis criteria for primary insomnia; the subject needed to have had in the past year trouble falling or staying asleep or not feeling rested waking up for at least one month; this had to significantly interfere with the routine activities; also any medical condition , which may cause this similar status, was excluded. If the subjects were negative or positive for the DSM-IV diagnostic criteria for generalized anxiety disorder or major depression that was annotated. Other mental disorders were excluded. Further, an average sleep time in the past year of less than four hours per night was considered insomnia. Of the very few patients with insomnia on sleep medication, we considered only the average sleep hours before starting the medication, independently on when the medication was started. Primary insomnia was present in 42.20% of families.The phenotype of insomnia is described as unknown if data are lacking. We could not collected insomnia data for all of the members in our 200 Italian families.IVS3-PSMD9 region containing the SNPs rs74421874 (IVS3+nt460 G>A), rs3825172 (IVS3+nt437 C>T) and the exon 5-coding region, containing the rs1043307/rs14259 (E197G A>G). We directly sequenced the PCR products, post-purification via EXOSAP-IT, on an automated ABI 3730 Sequencer.Using polymerase chain reaction (PCR) and with specific primers, we amplified in all family members the PSMD9 SNPs rs74421874 (IVS3+nt460 G>A), rs3825172 (IVS3+nt437 C>T) and rs1043307/rs14259 (E197G A>G) for linkage with primary insomnia and average sleep hours per night. Both non-parametric and parametric-linkage analysis for the qualitative phenotype were performed for the three SNPs, using Merlin softwareIn the 200 Italians families we tested the PSMD9 SNPs rs74421874 (IVS3+nt460 G>A), rs3825172 (IVS3+nt437 C>T) and rs1043307/rs14259 (E197G A>G) are in strong linkage disequilibrium (LD)We previously reported that the In order to rule out the effect of depression or anxiety on insomnia, we also performed a likelihood ratio test of each SNP association with insomnia considering anxiety or depression as a covariate, by using a mixed effects logistic regression model taking into account the families as a random effect factor in the model. For the Chi-square statistics, a corresponding p-value less than 0.05 means that the SNP tested has a significant effect on insomnia in the presence of the tested covariate and families as a random effect. The p-value is calculated by performing a likelihood ratio test, where the test statistic is the Chi-square statistic, which has a degree of freedom “DF” depending on the covariate and random effects.The following parameters were used for the parametric-linkage analysis based on the SNPs cluster, thus eliminating the LD inflation of the linkage signal, as it considers the SNPs all in the same locus: cluster haplotype disease-allele frequency 0.25; dominant model with penetrance for homozygous non-risk allele 0.13 , for heterozygous risk allele 1.00, for homozygous risk allele 1.00; recessive model with penetrance for homozygous non-risk allele 0.13, for heterozygous risk allele 0.13, for homozygous risk allele 1.00; additive model with penetrance for homozygous non-risk allele 0.13, for heterozygous risk allele 0.45, and for homozygous risk allele 0.90. Merlin computed the lod score from all the informative families for the insomnia phenotype (n = 28).For each significant non-parametric test performed, we calculated how many times similar p-values were expected by chance in 1,000 replicates of simulations using the gene-dropping method in order to exclude the presence of any false positives in our results. This analysis replaces real data with simulated data, while maintaining the pedigree structure, allele frequencies, and recombination fraction. These datasets were generated under the null hypothesis of no linkage.The quantitative-trait linkage analysis and variance-component analysis for the quantitative trait of average sleep hours per night was performed using MerlinAll results are reported as LOD scores as calculated by Merlin.PSMD9 SNPs rs74421874 (IVS3+nt460 A>G), rs3825172 (IVS3+nt437 C>T) and rs1043307/rs14259 (E197G A>G) are linked to primary insomnia via the non-parametric model .Our analysis shows that the PSMD9 SNPs to primary insomnia by using the LD-based model analysis and by testing each single SNP independently. The simulation analyses of 1,000 replicates exclude false positives and establish the data validity. The result of the non-parametric linkage analysis for primary insomnia are reported in Additionally, we identified a positive non-parametric linkage of the above-mentioned PSMD9 SNP rs74421874 (IVS3+nt460G>A) and SNP rs3825172 (IVS3+nt437C>T), independently, with insomnia using anxiety as covariate , but not using depression as covariate . The Chi-square statistics also shows a significant association of rs1043307/rs14259 (E197G A>G) with insomnia using anxiety as covariate  as well as using depression as covariate , rs3825172 (IVS3+nt437 C>T) and rs1043307/rs14259 (E197G A>G) studied are linked to primary insomnia in the Italian T2D families; it shows a trend towards linkage to sleep hours, with a trait heritability estimated to be circa 11%, and gene-trait heritability circa 25%. The different types of non-parametric-linkage analyses performed determined that the linkage is present independently from the presence of LD among variants; the simulations excluded the possibility that the results are due to random chance.Our study demonstrates that the PSMD9 coding variant rs14259 (E197G A>G) for a significant association with insomnia, considering anxiety or depression as a covariates. The two PSMD9 intronic SNPs rs74421874 (IVS3+nt460 G>A) and rs3825172 (IVS3+nt437 C>T) are significantly associated with insomnia considering anxiety as covariate, but lose significance in the association test with insomnia considering depression as covariate. Thus, the PSMD9 intronic variants rs74421874 (IVS3+nt460 G>A) and rs3825172 (IVS3+nt437 C>T) may provide vulnerability to the missense coding variant rs1043307/rs14259 E197G, which by itself may be the pathogenic variant.The logistic regression model supports the role of the In addition, we identified the recessive model as the most significant for the linkage, as our families’ structure contain sib-pairs, which are the most statistically powerful family dataset to identify recessive disorders.Our study may have also captured the linkage signal due to the presence of T2D; however, this risk is always present if we test for linkage in a complex disorder which is associated with another common disorder. Nevertheless, the variance-component analysis of sleep hours, based on a quantitative trait independent from the T2D qualitative phenotype, shows at least a trend towards linkage of sleep hours.PSMD9 is a ubiquitously expressed gene, which is implicated in anti-depressant therapy response and depression2435As insomnia is a risk factor for T2D, it is reasonable to anticipate that a gene may be linked to both insomnia and T2D. Furthermore, The IL6 receptor has been identified as a novel PSMD9-interacting partner which further supports PSMD9 role in the inflammatory responsePSMD9 is linked to primary insomnia in our T2D Italian families. Specifically, the PSMD9 coding variant rs1043307/rs14259 E197G is significantly associated with insomnia taking into account anxiety or depression as covariate, while the PSMD9 intronic SNPs rs74421874 (IVS3+nt460 G>A) and rs3825172 (IVS3+nt437 C>T) remain significantly associated with insomnia only when taking into account anxiety –and not depression– as covariate. These findings are new, and should be independently replicated in other populations. These data, if confirmed in other populations, imply that PSMD9-targeted therapies may prevent or treat primary insomnia, depression, and T2D, and/or to manage personalized therapy based on patient genotypes.In summary, How to cite this article: Hao, H. et al. T2D and Depression Risk Gene Proteasome Modulator 9 is Linked to Insomnia. Sci. Rep.5, 12032; doi: 10.1038/srep12032 (2015).


The electrogenic Na/Ca exchanger (NCX) mediates bidirectional Ca movements that are highly sensitive to changes of Na gradients in many cells. NCX1 is implicated in the pathogenesis of heart failure and a number of cardiac arrhythmias. We measured NCX1 palmitoylation using resin-assisted capture, the subcellular location of yellow fluorescent protein–NCX1 fusion proteins, and NCX1 currents using whole-cell voltage clamping. Rat NCX1 is substantially palmitoylated in all tissues examined. Cysteine 739 in the NCX1 large intracellular loop is necessary and sufficient for NCX1 palmitoylation. Palmitoylation of NCX1 occurs in the Golgi and anchors the NCX1 large regulatory intracellular loop to membranes. Surprisingly, palmitoylation does not influence trafficking or localization of NCX1 to surface membranes, nor does it strongly affect the normal forward or reverse transport modes of NCX1. However, exchangers that cannot be palmitoylated do not inactivate normally  and do not promote cargo-dependent endocytosis that internalizes 50% of the cell surface following strong G-protein activation or large Ca transients. The palmitoylated cysteine in NCX1 is found in all vertebrate and some invertebrate NCX homologs. Thus, NCX palmitoylation ubiquitously modulates Ca homeostasis and membrane domain function in cells that express NCX proteins.—Reilly, L., Howie, J., Wypijewski, K., Ashford, M. L. J., Hilgemann, D. W., Fuller, W. Palmitoylation of the Na/Ca exchanger cytoplasmic loop controls its inactivation and internalization during stress signaling. Cardiac diastole relies on effective removal of Ca from the cytoplasm of ventricular myocytes to allow relaxation and refilling of the ventricles. Removal of Ca occurs as a balanced competition between sarcoplasmic reticulum Ca ATPases SERCA 2a) and Na/Ca exchangers; Ca pumps sequester Ca into the sarcoplasmic reticulum, and Na/Ca exchangers (NCXs) extrude Ca to the extracellular space . In larga and Na/Contractile abnormalities that occur in left ventricular hypertrophy and failure can be related to an imbalance between SERCA and NCX. Most commonly, depletion of intracellular Ca stores occurs because either SERCA activity is decreased or NCX activity is increased , 6. Curr14via 2 Ca binding domains (CBDs)  domain: PIP2 activates NCX mostly by antagonizing Na-dependent inactivation  , 24. Bots (CBDs) , 26] andtivation , 28. Thetivation –31, and tivation .via a thioester bond between a cysteine sulfhydryl side chain and the fatty acid palmitate  commonly regulates membrane association, subcellular location, trafficking, turnover rate, and enzymatic activity of both peripheral and integral membrane proteins -dependent endocytosis that can be activated by G proteins and large Ca transients.The reversible acylation of proteins proteins . The actproteins . Here, wAll experiments involving animals were approved by the University of Dundee Welfare and Ethical Use of Animals Committee.Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) and maintained by The University of Iowa, Department of Biology .Anti-NCX1 was from Swant, anti-caveolin 3 and flotillin 2 were from BD Biosciences , and anti–green fluorescent protein was from Abcam . The monoclonal antibody α6F raised against the sodium pump α1 subunit by Douglas M. Fambrough  was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the Palmitoylated proteins were purified using thiopropyl Sepharose in the presence of neutral hydroxylamine after alkylation of free thiols with methyl methanethiosulfonate, as described previously . UnfractRepresentative fractions of cell surface proteins were prepared by briefly treating cells with 1 mg/ml sulfo-NHS-SS-biotin  for 10 min at 37°C to biotinylate integral surface membrane proteins with extracellular primary amines, which were subsequently purified using streptavidin Sepharose .Canine NCX1.1 cDNA was kindly provided by Professor Godfrey Smith . DsRed-ER was from Clontech  and Grasp65-mCherry was kindly provided by Dr. Jon Lane . Yellow fluorescent protein (YFP) NCX1 large intracellular loop (YFP-NCX1-IC) was created by inserting canine NCX1.1 cDNA (codons 219 to 761) at the 3′ end of YFP in vector pEFYP-C1 (Clontech). All transfections of plasmid DNA used Lipofectamine 2000  according to the manufacturer’s instructions. Human embryonic kidney (HEK)-derived FT-293 cells expressing tet-inducible wild-type (WT) and C739A NCX1 were generated using the Invitrogen Flip-In T-Rex system.Ca-tolerant adult ventricular myocytes were isolated from the hearts of male Wistar rats (250–300 g) by retrograde perfusion of collagenase in the Langendorff mode. Human-induced pluripotent stem cells were differentiated into cardiac myocytes at the University of Dundee iPSC facility using established protocols.Images were acquired from cells fixed in 4% paraformaldehyde 24 h after transfection using a Leica SP5 confocal microscope. Acquisition parameters were : YFP, 519/542; DsRed, 575/644; mCherry, 580/700.FT-293 cells were removed from dishes by treatment with trypsin (0.1%) and were patch clamped (whole-cell configuration) at 37°C with pipette perfusion and fast bath solution exchange and capacitance measurements as described previously . HoldingCaveolin-enriched buoyant membranes were prepared from ventricular myocytes using a discontinuous sucrose gradient as described previously . FollowiMutagenesis reactions were performed with the Quikchange Lightning and Quikchange Multi Site-Directed Mutagenesis kits . Individual cysteines were mutated to alanines using the Quikchange Lightning Site-Directed Mutagenesis Kit (Agilent). The mutagenesis primer pairs used were as follows: C383A: forward, CAAGGGACCTATCAGGCTCTGGAGAACTGTGGG, reverse, CCCACAGTTCTCCAGAGCCTGATAGCTCCCTTG; C387A: forward, CAGTGTCTGGAGAACGCTGGGACTGTAGCCC, reverse, GGGCTACAGTCCCAGCGTTCTCCAGACACTG; C383A/C387A: forward, GGGACCTATCAGGCTCTGGAGAACGCTGGGACTGTAGCC, reverse, GGCTACAGTCCCAGCGTTCTCCAGAGCCTGATAGGTCCC; C485A: forward, GTTTCTGCGCTCGCTGCCCTGGGATCTCCC, reverse, GGGAGATCCCAGGGCAGCGAGCGCAGAAAC; C557A: forward, GGACTTTGAGGACACTGCTGGAGAGCTCGAATTCC, reverse, GGAATTCGAGCTCTCCAGCAGTGTCCTCAAAGTCC; C731A: forward, GATGATGACGACGATGAAGCTGGAGAGGAGAAGCTG, reverse, CAGCTTCTCCTCTCCAGCTTCATCGTCGTCATCATC; C739A: forward, GGAGAAGCTGCCCTCCGCTTTCGATTATGTGATGC, reverse, GCATCACATAATCGAAAGCGGAGGGCAGCTTCTCC; C914A: forward, CAAGCTCCTCACATCCGCCCTCTTCGTGCTCCTATG, reverse, CATAGGAGCACGAAGAGGGCGGATGTGAGGAGCTTGG.A cys-less NCX1 intracellular loop  was created using the Quikchange Multi Site-Directed Mutagenesis Kit, which requires forward primers only. The same mutagenesis reaction also yielded all other mutants described.Analysis of the rat neural palmitoyl proteome has previously identified NCX1 and NCX2 as being palmitoylated , but didTo identify the site of palmitoylation in NCX1, individual cysteines predicted to reside in or close to the cytoplasmic domains , membrane (Triton X-100 soluble), and insoluble fractions. Cys-less YFP-NCX1-IC was entirely cytosolic . Second, in the absence of intracellular ATP, a large Ca transient inactivates NCX1 by causing depletion of PIP2, through the activity of PLCs (P < 0.01). We therefore conclude that palmitoylation facilitates and may be required for complete inactivation of NCX1 by both removal of cytoplasmic Ca and by loss of PIP2.NCXs contribute a substantial proportion of Na load in cardiac muscle, and Na-dependent inactivation of NCXs may decrease myocyte Na load during metabolic stress as part of a cell program to shift Ca turnover to internal recycling and away from turnover across the sarcolemma . We meas of PLCs . In this of PLCs B. This nP < 0.01) to about 30% of peak currents.As a means to cause inactivation of NCX1 without changing cytoplasmic free Ca, we included polylysine in ATP-free cytoplasmic solutions to bind anionic phospholipids prior to activation of exchange current by Ca . Figure 3+ to bind to PIP2 head groups with extraordinarily high affinity, thereby masking PIP2 head groups in a specific manner , and currents obtained in C739A-expressing cells are 4-fold greater . Therewith, results from 4 different experimental protocols concur that inactivation of NCX1 is decreased by the C739A mutation.Finally, we exploited that fact that EGTA/aluminum chelates effectively provide Alc manner . Using AP < 0.01). This form of endocytosis is strongly cargo-dependent, as illustrated by the near absence of endocytosis in FT-293 cells expressing no exchangers  with high (<1 µM) affinity activates the exchanger, and Ca binding to CBD2 (residues 492–592) with lower affinity relieves Na-dependent inactivation . The extent of Na-dependent inactivation also varies significantly in different experimental models . This effect of palmitoylation on lipid regulation of an ion transporter bears similarities to the regulation of the sodium pump by its palmitoylated accessory subunit phospholemman, which appears to rely on palmitoylation of phospholemman modulating regulatory lipid interactions with the pump (2, as well as other phospholipids (The presence of a membrane anchor at position C739 can be expected to influence the tilt of multiple interacting transmembrane domains in NCX1 . Transitd groups  and 6 al minimal . Hence,  minimal . That sathe pump , 64. A lholipids , and a sholipids . As the In addition to modulating the immediate interactions of NCX1 transporters with phospholipids, our results indicate that palmitoylation of NCX1 promotes its participation in membrane domains that can be internalized in a cargo-dependent manner without involvement of classic endocytic proteins . These rNCX1 is a member of the cation/Ca exchanger superfamily, which is defined by the presence of highly conserved α-repeat regions within 2 membranous hydrophobic domains, which are separated by a hydrophilic cytosolic loop rich in acidic amino acids. This superfamily of 147 transporters from both eukaryotes and prokaryotes consists of 5 distinct protein families . We usedDrosophila exchanger Calx, but it is only present in NCX2 from Caenorhabditiselegans. We therefore suggest that this palmitoylation site is an adaptation in vertebrates and higher invertebrates to regulate NCX activity.Alignment of NCX family members B indicatAlthough palmitoylation of NCX1 in its large intracellular loop occurs in the secretory pathway, it is surprisingly not required for NCX trafficking or function in our HEK overexpression system. Palmitoylation regulates NCX1 inactivation, possibly influencing the ability of the XIP region to inhibit exchanger activity, and palmitoylation promotes the participation of NCX1 in membrane domains that can be internalized in a cargo-dependent fashion.


AR) gene, located on the X chromosome, is not known to be involved in sex determination. In the Japanese frog Rana rugosa the AR is located on the sex chromosomes . Phylogenetic analysis shows that the AR on the X chromosome (X-AR) of the Korean R. rugosa is basal and segregates into two clusters: one containing W-AR of Japanese R. rugosa, the other containing Y-AR. AR expression is twice as high in ZZ  compared to ZW  embryos in which the W-AR is barely expressed. Higher AR-expression may be associated with male sex determination in this species. To examine whether the Z-AR is involved in sex determination in R. rugosa, we produced transgenic (Tg) frogs carrying an exogenous Z-AR. Analysis of ZW Tg frogs revealed development of masculinized gonads or ‘ovotestes’. Expression of CYP17 and Dmrt1, genes known to be activated during normal male gonadal development, were up-regulated in the ZW ovotestis. Testosterone, supplied to the rearing water, completed the female-to-male sex-reversal in the AR-Tg ZW frogs. Here we report that Z-AR is involved in male sex-determination in an amphibian species.In mice and humans, the androgen receptor ( R. rugosa (2n = 26) has two sex-determining systems within one species R. rugosa are found in the Korean R. rugosaR. rugosa evolved through two independent inversions on chromosome 7 R. rugosa.Sex is genetically determined in most vertebrates. As in other vertebrate species, heterogametic sex chromosomes in amphibians determine the male (XX/XY) or female (ZZ/ZW) fate AR-knockout mice AR knock-in mice AR does not participate in sex determination in mice. The Sry on the Y chromosome is recognized as the master genetic determinant of male fate in this species Androgens exert a variety of effects in target tissues such as male reproductive organs, brain and skeletal tissues. Androgenic effects are mediated by tissue-specific transcriptional control of target genes via nuclear androgen receptor (AR) R. rugosa the AR gene is located on the sex  chromosomes and reportedly on the inverted region of the Y and W chromosomes AR gene is in the process of evolutional degradation from lack of recombination between the inverted and non-inverted regions of the sex chromosomes . In addition, it has been proposed that degradation of the W-AR began just after or at the origin of the ZW sex-determining system in R. rugosaW-AR expression levels are extremely low in R. rugosa embryos, perhaps owing to promoter sequence variation and cognate transcription factor interaction between the W- and Z-AR promoters W-AR is transgenically expressed as demonstrated in reporter assays using Xenopus laevis kidney-derived A6 cells R. rugosa W-AR is still in an early phase. During sex determination, R. rugosa male gonads synthesize more androgens than females AR expression is up-regulated in the male gonad of R. rugosa tadpoles prior to sex determination AR may be involved in male sex determination in this species. To test this hypothesis, we produced transgenic (Tg) frogs carrying an exogenous Z-AR driven by the promoter region of both Z-AR and EF1α genes. Strikingly, a subset of the Z-AR-Tg female (ZW) frogs formed composite gonads or “ovotestes”. Here we report that the Z-AR is involved in male sex determination in a vertebrate species.In All the animal experiments in this study were performed in respect of the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions  and the Prevention of Cruelty to Animal Act . This included official approval from the Committee of Animal Experimentation of Waseda University (Permit Number: 2013-A005).R. rugosa frogs were used in this study. Unfertilized eggs were artificially ovulated and inseminated Female heterogametic (ZZ/ZW) AR using primers shown in AR gene tree was constructed using the UPGMA method [Genetyx Mac version 14.0.9].To construct a phylogenetic tree of the AR gene, we employed PCR to amplify the promoter region of the Z-AR cDNA (pink bar) and the promoter of Z-AR (blue arrow) into the p(I-SceI)DPCG construct  into the pT2AL200R150G (prrT2ARG). First, we amplified the Z-AR cDNA by PCR using a pair of primers specific for the nucleotide sequence of Z-AR cDNA: forward 5′-AAAAAAGATATCATGGAGGTGCACATTGGACT-3′ and reverse 5′-GAGATGAAGGTGTCAAGTACCTATAGAAAAAA-3′, and the R. rugosa Z-AR (DDBJ Accession No. AB491761) or EF1α (AB822986) promoter region. Then we inserted the Z-AR  or EF1α  promoter region ligated to the 2,312-bp Z-AR cDNA into the p(I-SceI)DPCG vector between the SalI/Agel sites and the pT2AL200R150G vector SalI/XhoI sites, respectively. Each AR expression vector carried either the promoter of the AR gene or of EF1α. V5 (yellow bar) represents the small epitope tag polypeptides (GKPIPNPLLGLDST) of the V5 protein from the paramyxovirus of simian virus 5 (SV5). Restriction enzyme sites for I-SecI and TolII recognition sites are indicated by black arrowheads.To construct the vectors for transgenesis we inserted the 2.3-kbp  pARPAR,  [18], orI-SceI meganuclease- I-SceI meganuclease (NEB) and the I-SceI-cleaved plasmid encoding Z-AR and V5, and the Tol II mRNA Z-AR integration into genomic DNA, we extracted DNA from the tail tip of all Tg frogs just after metamorphosis, using the AllPrep DNA/RNA Micro Kit (QIAGEN). The PCR primers used were: forward, 5′-GCGGTTTTTCCAACTTACCA-3′ and reverse, 5′-CGAGACCGAGGAGAGGGTTA-3′).To produce Tg frogs, we used the Z-AR/V5 expression in Tg ZW gonads. Total RNA was prepared from the gonads of Wt and Tg frogs just after metamorphosis using ISOGEN (NIPPON GENE) and cDNAs were synthesized Z-AR/V5, CYP17A1, and Dmrt1) or 25 cycles (GA3PDH) of 95°C (30 sec), 62°C (30 sec), and 72°C (1 min), ending with 7 min of extension at 72°C. DNA fragments for Z-AR/V5 (260-bp), CYP17A1 , Dmrt1  and GA3PDH  cDNAs were amplified by PCR using a set of primers for each respective template. Primer sequences are given in We employed PCR analysis to examine Z-AR cDNA (AB372103) encoding polypeptide RVISCKRNNPASSSRRFFQL (AR20) corresponding to residues 695–714, and the other the CYP17A1 cDNA (AB284119) encoding polypeptide DEKEWVNPHLFNPDRFLDENGNRVYS (CYP1726), corresponding to residues 409–434. The DNA fragments were inserted into the BamHI/XhoI sites of the pGEX-4T expression vector . The GST-AR20 and GST-CYP1726 expression cassettes were transfected into E. coli BL21. The transfected cells were sonicated in ice-cold water using an Ultrasonic Processor . We purified GST-AR20 and GST-CYP1726 proteins according to the manufacturer's protocol  and immunized 8-week-old BALB/c female mice three times with 50 µg of protein in complete Freund's adjuvant (Wako) at 2-week intervals. Sera were collected and tested 5 days after the last booster injection and used for immunohistochemistry.For production of mouse antibodies, we designed two sets of primers. One pair amplified the R. rugosa adult testes protein (30 µg) and molecular weight markers (BIO-RAD) were subjected to electrophoresis using a 10% polyacrylamide gel and 30 mA for 1 to 2 hrs R. rugosa adult testes.The primary antibodies against Vasa Gonads were surgically excised from Wt and Tg frogs just after metamorphosis, fixed and frozen as previously described Images were scanned and adjusted for brightness and contrast by Adobe Photoshop CS2.R. rugosa is found in the Korean R. rugosaW-AR gene was inherited from the ancestral type X-AR of the Korean R. rugosa, we constructed an AR gene tree using the UPGMA method [Genetyx Mac version 14.0.9], based on the nucleotide sequences of the promoter region of the AR in the 6 local populations. The X-AR in the Korean population appeared to be basal and segregated into two clusters: one contained the X-AR of western Japan, the Z-AR northern Japan including Sado island and the Y-AR of the central population; the other contained the X-AR of eastern and central Japan and the W-AR of the northern population  testis. Wt female (ZW) and male (ZZ) frogs developed normal bilateral ovaries and testes, respectively   (Tg ZW frogs carrying the exogenous  145 nM) . Many ge 145 nM) . Similar 145 nM) .To examine the internal structure of non-Tg and Tg gonads, tissue sections were immunostained for laminin, a marker of the basement membrane AR driven by the Z-AR promoter (pARPAR) into 450 fertilized eggs of R. rugosa. Two hundred and thirty injected eggs (51.1%) developed into tadpoles at St. I (limb buds are visible) and 222 tadpoles (49.3%) grew into frogs just after metamorphosis, of which 111 were characterized  developed an intermediary gonad, a hybrid of testis and ovary, called ‘ovotestis’. These hybrid organs were classified into the three categories mentioned above .We introduced an exogenous cterized . In the AR-Tg ZW frogs formed testes.When T was present in the rearing water, 35 tadpoles metamorphosed into frogs. All 14 of the Wt male (ZZ) frogs developed testes, as expected. All 12 of the Wt ZW frogs and all 9 of the AR driven by the R. rugosa EF1α promoter (prrT2ARG) into 200 fertilized eggs  of Type 3, but not the Tg left gonad  frogs formed ovotestes  frog formed ovotestes co-expressing AR and GFP. Hence, it is reasonable to conclude that Z-AR has become a gene involved in sex determination in R. rugosa possessing the ZZ/ZW sex-determining system. To the best of our knowledge, this is the first report showing that AR-transgenesis induces female-to-male sex-reversal in a species of vertebrate.Dmrt1 and CYP17 expression is enhanced in the XX sex-reversed gonad as well as in the normally differentiating testis of R. rugosaDmrt1 expression is up-regulated during the late stages of male sex differentiation Dmrt1 in XX tilapia fish results in partial to complete sex reversal Dmrt1 expression in the ZW ovotestes are compatible with these previous findings. Furthermore, we found that the Type 3 Tg gonad, the masculinized part (the ovotestis) that expressed the exogenous AR also expressed Dmrt1 and CYP17, while the non-masuculinized part (the ovary) did not express any of these 3 genes   . HoweverAR when T is supplied. In the ZW testis, Dmrt1 and CYP17 mRNA levels increase, as they do in the developing ZZ testis, indicating that T regulates Dmrt1 and CYP17 expression directly or indirectly through the AR. Threshold levels of T and AR in the ZW gonads required for sex determination have yet to be determined; amounts available may be insufficient to trans-activate cognate genes, or to initiate the gene cascade necessary for specification of the male fate. However, a ZW gonad would be expected to mature into testes if the combined levels of T and AR were sufficient for testis formation. In support of this is the finding that more androgens are synthesized in R. rugosa Wt ZZ gonads during sex determination than in Wt ZW gonads Z-AR can induce sex-reversal in ZW R. rugosa, suggesting that androgen and its receptor are involved in non-genotypically programmed male sex determination in this species, and moreover, this report provides evidence for the first time that evolutionary degradation of the W-AR has led to a critical role for the AR (Z-AR) in sex determination in a species of vertebrate.ZW female frogs can develop testes in the absence of Tg Figure S1Histology of the type 3 gonads. All 3 of the Type 3 Tg ZW gonads obtained in this study are shown . Dashed lines indicate the borders of the gonads.(TIF)Click here for additional data file.Figure S2AR expression in Wt ZW (+T) gonads. The PCR analysis was performed to examine Z-AR expression in Tg and Wt ZW gonads treated with (+) or without (−) T as described elsewhere Z-AR and GA3DPH expression are shown in Z-AR and GA3PDH mRNA level in the gonad, respectively.(TIF)Click here for additional data file.Figure S3Immunohistology of Wt ZW (+T) gonads. Frozen sections from the Wt ZW (+T) testis were stained with the antibodies of AR, CYP17, Vasa and laminin (Lam), and counterstained with DAPI and also HE. AR- and CYP-17 positive signals were observed in the Wt ZW testis treated with T.(TIF)Click here for additional data file.


Dear Editor,Hearing loss is one of the major public health problems. The mitochondrial DNA mutations has been found to be associated with both aminoglycoside-induced and nonsyndromic hearing loss . Furthermore, according to their distinct clusters of mtDNA polymorphisms, we found the mtDNAs of 11 pedigrees belonged to 4 common eastern Asian mtDNA haplogroups A, B, D and F.OR = 2.10; 95% CI = 1.38–3.25; P < 0.01). Furthermore, the increased frequency of 1555A>G mutation on haplogroup D background is attributable largely to specific subhaplogroup D5 . In contrast, those families on haplogroup M8 and G background tended to have a reduced frequency of 1555A>G mutation (P < 0.05).Besides the 11 families with hearing loss above, we collected additional 131 pedigrees carrying 1555 A>G mutation among eastern Asian population from previous publications for further analysis. Of the 142 mtDNAs analyzed, 74 were categorized into macro-haplogroup M, and the rest 68 pedigrees’ mtDNAs were assigned to macro-haplogroup N. mtDNA haplogroup distribution analysis showed that 1555A>G mutation was widely dispersed among 11 major Eastern Asian haplogroups D, M7, M8, M10, M11, G, F, B, R, A and N9 . This observation strongly indicated that the 1555A>G mutation arising independently in different mtDNA haplogroup backgrounds among eastern Asian populations. As shown in Table P = 0.006). Furthermore, when the aminoglycosides-exposed hearing loss cases were excluded, these haplogroup B pedigrees still showed significantly higher penetrance of hearing loss symptom .To further understand the role of mtDNA haplogroup on phenotypic expression for 1555A>G mutation, we evaluated the penetrance of hearing loss among 142 pedigrees (Table S3). A wide range of the penetrance of hearing loss were observed among the 142 pedigrees, with the average of 33.8% and 21.8% when aminoglycosides-induced hearing loss was included or excluded, respectively. Then the clinical penetrance of 1555A>G mutation on specific haplogroup were compared with that on all mtDNA backgrounds


The economically important DT2008 and the model Williams 82 (W82) soybean cultivars were reported to have differential drought-tolerant degree to dehydration and drought, which was associated with root trait. Here, we used 66K Affymetrix Soybean Array GeneChip to compare the root transcriptomes of DT2008 and W82 seedlings under normal, as well as mild (2 h treatment) and severe (10 h treatment) dehydration conditions. Out of the 38172 soybean genes annotated with high confidence, 822 (2.15%) and 632 (1.66%) genes showed altered expression by dehydration in W82 and DT2008 roots, respectively, suggesting that a larger machinery is required to be activated in the drought-sensitive W82 cultivar to cope with the stress. We also observed that long-term dehydration period induced expression change of more genes in soybean roots than the short-term one, independently of the genotypes. Furthermore, our data suggest that the higher drought tolerability of DT2008 might be attributed to the higher number of genes induced in DT2008 roots than in W82 roots by early dehydration, and to the expression changes of more genes triggered by short-term dehydration than those by prolonged dehydration in DT2008 roots vs. W82 roots. Differentially expressed genes (DEGs) that could be predicted to have a known function were further analyzed to gain a basic understanding on how soybean plants respond to dehydration for their survival. The higher drought tolerability of DT2008 vs. W82 might be attributed to differential expression in genes encoding osmoprotectant biosynthesis-, detoxification- or cell wall-related proteins, kinases, transcription factors and phosphatase 2C proteins. This research allowed us to identify genetic components that contribute to the improved drought tolerance of DT2008, as well as provide a useful genetic resource for in-depth functional analyses that ultimately leads to development of soybean cultivars with improved tolerance to drought. Glycine max L.) has been regarded as one of the major legume crops worldwide with multibillion dollars in value. Its seed product provides a substantial source of vegetable protein and oil, micronutrients and minerals for animal feed and human consumption  the transcriptome-wide changes in soybean dehydrated roots vs. non-dehydrated roots and (ii) analyze the genome-wide differential gene expression in the root tissues of Williams 82 (W82) and DT2008, which have differential dehydration/drought-responsive phenotype , 2 (early stress), and 10 h (late stress) of dehydration Figure  by microhttp://phytozome.jgi.doe.gov/pz/portal.html). Using this latest Glyma v2.0 annotation, the 66K soybean GeneChip allowed us to study the expression of 38172 genes with high confidence. These genes were subjected to a search for differentially expressed genes (DEGs) using the criterion of two-fold expression change (q < 0.05) ] released by Phytozome 10.1  , RLK (receptor-like), and MAP (mitogen-activated protein) kinases and PP2C (protein phosphatase 2C) proteins based on sequence homology with their To study the correlation between the differential gene expression in roots of W82 and DT2008 and their differential drought tolerance, we first compared their root transcriptomes under both normal and dehydration conditions. With regard to the upregulated gene sets derived from DT2008 vs. W82 comparison, we found that under well-watered conditions, 82 genes were upregulated in DT-C/W-C comparison, whereas under dehydration, a total of 147 genes were upregulated in DT-D/W-D comparison, with more induced genes being identified during earlier stress Figure . Namely,Glyma.04G083000, was upregulated in both dehydrated W82 and DT2008 roots   and DT-D/W-D (dehydration conditions) comparisons, respectively. We also observed that more genes (194 vs. 26 genes) were downregulated in DT-D/W-D comparison by the short-term 2 h (DT-D2/W-D2) rather than the prolonged 10 h (DT-D10/W-D10) dehydration treatment Figure .In a similar manner, the downregulated gene sets obtained from the comparative analysis of root transcriptomes of W82 and DT2008 under non-stressed and stressed conditions (DT-C/W-C and DT-D/W-D) were also analyzed to identify dehydration-repressible genes exhibiting lower expression in drought-tolerant DT2008 as these genes would also be responsible for better performance of DT2008 relative to W82 under drought. Thus, downregulated gene sets of DT-C/W-C, DT-D/W-D, W-D/W-C, and DT-D/DT-C comparisons were evaluated by a Venn analysis as well , we were able to examine the expression of 38172 genes with high confidence through our transcriptome analysis  and W82 (W-D10/W-C vs. W-D2/W-C) by 10 h than 2 h dehydration treatment Figure . These dNAC genes, exhibited higher expression level in DT2008 roots than W82 roots, especially under well-watered and early dehydration treatment  , glyoxalase I family protein and xyloglucan endotransglycosylase, may play important roles in better adaptation of DT2008 to drought relative to W82 as supported by previous studies  Table . This ge2) Table . ModulatGlyma.02G069400 codes for an ortholog of Arabidopsis BIN2 (brassinosteroid-insensitive 2) (Table Glyma.02G069400 might therefore contribute to increased drought tolerance of DT2008. Another example is Glyma.12G202400 encoding a protein with high homology to an Arabidopsis phosphatase 2C protein (AT4G03415) (Table 2+-dependent protein kinase/sucrose non-fermenting related kinase (CPK/SnRK) superfamily , there have been published reports that several bZIP TFs act as negative regulators of drought tolerance. For instance, overexpression of OsbZIP52 in rice significantly enhanced sensitivity of transgenic plants to cold and drought stresses  Table . It was 2) Table . Repressns Table . Severalns Table . As for In summary, our comparative analysis of root transcriptomes of DT2008 and W82 under both well-watered and dehydration conditions have allowed us to identify genetic components that might contribute to the improved drought tolerance of DT2008. Our study also provides a useful genetic resource for scientists with interests in basic and/or applied research to carry out further in-depth gene characterization and functional analyses. This in turn will contribute to deeper understanding of mechanisms regulating drought responses and adaptation in soybean, which ultimately leads to development of soybean cultivars with improved tolerance to drought.−2 s−1 photon flux density). For the collection of well-watered and dehydrated root tissues, 14-d-old soybean plants with two trifoliate leaves (V2 stage) were carefully removed from pots, then gently washed to remove soil from the roots. Subsequently, the W82 and DT2008 plants were dried on a filter paper for different time periods under the condition of 44% relative humidity, 23°C room temperature and 10 μmol m−2 s−1 photon flux light intensity. The severity of the stress level was measured by determination of RWC of the aerial parts of dehydrated plants. After the dehydration treatment, plants dehydrated for 0, 2, and 10 h were collected and the roots were separated from the shoots. Root samples were quickly frozen in liquid nitrogen and stored at −80°C until RNA purification. Accordingly, the following root samples were collected in three biological replicates from W82 and DT2008 plants for microarray analysis: W82 well-watered control 0 h (W-C), W82 dehydrated 2 h (W-D2), W82 dehydrated 10 h (W-D10), DT2008 well-watered control 0 h (DT-C), DT2008 dehydrated 2 h (DT-D2); DT2008 dehydrated 10 h (DT-D10).W82 and DT2008 soybean plants were separately grown in pots containing vermiculite (6 plants per 6-liter pot) under well-watered conditions in a controlled greenhouse  was estimated by a Student's t-test, while its certainty level  was assessed using Benjamini and Hochberg False Discovery Rate. Genes with expression change ≥2-fold (q < 0.05) were regarded to be differentially expressed. The obtained microarray data have been deposited in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/browse/?view=series) (accession number GSE65553)RNAs were extracted from root samples using the Trizol reagent  as recommended by the manufacturer's protocol. Purified total RNA was subsequently subjected to a DNase I treatment prior to the quality assessment by an Agilent 2100 Bioanalyzer  was used to annotate and analyze the microarray data as according to previously published methods (Thimm et al., MapMan (Several genes were randomly selected for verification of the microarray data using RT-qPCR.Fbox gene was used as a reference gene in the RT-qPCR analysis of RNA samples from three biological replicates (Le et al., The specific primer pairs used in RT-qPCR were listed in Supplementary Table L-SPT conceived research and wrote the manuscript. CVH, YW, UTT, DTL, MT, and KHN performed the experiments and analyzed the data. MS and DVN contributed research materials.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins.Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1’s function remained obscure; inherent dynamics of SSBs and PARP-1’s multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1’s signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodifcation in   •We identify the structural basis of DNA single-strand break detection by PARP-1•An NMR/X-ray approach reveals how multi-domain allostery underlies activity switch•The molecular model of PARP-1 assembly couples damage detection to PAR signaling•cis releases enzyme so DNA repair and replication can proceedAutomodification in  Eustermann et al. reveal the structural basis for recognition of DNA single-strand breaks by human PARP-1 and how this drives a multi-domain folding cascade to operate the allosteric switch initiating DNA damage signaling. The structural model of PARP-1 on a DNA break helps rationalize inhibitor action. It is the founding member of a family of enzymes that modify a wide variety of target proteins with poly(ADP-ribose) (PAR), a highly negatively charged, branched-chain posttranslational modification derived from NAD+ . Acute D+  and link+ . Excessiepletion , whereasepletion . PARP-1 epletion . Most prepletion . Numerouepletion . These iepletion , and posepletion . Howeverepletion  has beenDNA single-strand breaks (SSBs) are by far the most frequent form of DNA damage, resulting both directly from oxidative damage and as intermediates in other DNA repair pathways . AlthougHere we used an integrated nuclear magnetic resonance (NMR)/X-ray approach to establish the molecular mechanism by which PARP-1 senses SSBs and becomes allosterically activated. Because key interactions are detected in solution using NMR, inherent flexibility no longer poses the obstacle that it does for crystallography, allowing us to interrogate complexes representing successive assembly states of the system and, thereby, build up the stepwise, co-operative, multi-domain folding pathway that underlies the operation of PARP-1’s DNA damage-dependent activity switch during genomic maintenance.To understand the molecular basis of the PARP-1 response to genomic damage, we first sought to dissect how DNA damage is recognized. While crystallographic studies so far have employed mimics of DSBs, we set out to determine the structural basis of SSB recognition using solution-state NMR spectroscopy. We used a minimal protein construct comprising PARP-1’s flexibly linked N-terminal zinc fingers F1F2 B, corresThe challenges of determining a structure of this size by NMR were overcome mainly by combining TROSY-based experiments  with a t2)  is the only one that triggers activation . Given tTaken together, our results identify a consensus that many DNA structures that activate PARP-1 have in common, comprising two flexibly linked DNA stems with exposed bases at the ends, independent of 3′ or 5′ modifications. When F2 has initiated recognition by binding at the 3′ stem, subsequent scanning for the second site by the flexibly linked F1 domain resembles a “fly-casting” mechanism , elegantMolecular Cell]). Intriguingly, there are no direct contacts in our model between the CAT domain and F1, F2, or the DNA, showing that communication must occur through F3 and WGR. Our mutational analysis shows that interactions of the latter domains within monomeric PARP-1 are essential for activation by SSBs  clearly revealed that SSB binding by F1F2 triggers interactions of F3 with both F1 and the 5′ stem of the DNA . However complex f and alsF3 W246A e, and, iF3 W246A C. The arF3 W246A D. Not onF3 W246A B, but, aF3 W246A C.By showing how high-affinity SSB detection by F1F2 provides the driving force to bring together F3 and WGR in the correct spatial orientation in the absence of CAT, we reveal intermediate steps on a multi-domain folding pathway that explain why CAT binding and consequent destabilization take place, initiating productive PAR catalysis. Association of each domain is required to create the binding platform for the next. Because individual interactions subsequent to DNA damage recognition are weak and are built from small parts on separate components, these must be pre-organized by previous assembly steps to form an organized whole . Our datcis, with the same PARP-1 molecule that detects DNA damage also serving as the substrate for automodification.The structural model of full-length PARP-1 represents the enzyme in a state primed for productive PAR catalysis. It remains an open question what specificity may exist in substrate selection. Proteomics studies have so far not yielded a defined consensus modification site , and PARtrans has been reported, based mainly on observed bimolecular reaction kinetics when active calf thymus DNA containing a highly diverse range of DNA ligands was used for activation  and DSB (single hairpin) mimics, PARP-1 automodification occurs almost exclusively in cis. Selective in cis modification likely presents specific regions of PARP-1 for automodification. Indeed, our structural model of SSB-bound full-length PARP-1 together with HXMS data . Well known examples include the nuclear hormone receptors  caused by PARP-1 inhibition leads to collapsed replication forks and an increase of toxic DSBs , whereasTo fulfill its functions, PARP-1 requires a combination of rapid response to an initial stimulus coupled with tight spatio-temporal control of catalytic activity, while also allowing more subtle degrees of regulation. The mechanism of SSB detection and allosteric activation we describe here is ideally suited to meet these demands. Ligand-induced multi-domain folding of domains within a single polypeptide chain represents an extremely efficient way to communicate a signal while maintaining, because of its dynamics, the potential for versatility and modulation so far more often associated with more complex but slower protein interaction networks. It seems likely that other signaling systems may have evolved similar solutions. Allosteric control of dynamic multi-domain proteins has emerged as one of the key molecular mechanisms underlying complex cellular signaling events, and the principles described here may well apply more generally.Full experimental procedures for cloning, expression, and purification of deuterated, back-labeled protein samples for NMR spectroscopy (including those having different isotope labeling patterns in different domains and prepared using sortase ligation), DNA preparation and purification, isothermal calorimetry, fluorescence anisotropy, analytical ultracentrifugation, activity assays, live-cell imaging, and NMR experiments and assignments are provided in the 3ODA for F1 and 3ODC for F2) showed that the structures were equivalent to approximately the degree expected for X-ray structures in this resolution range (2.6–2.8 Å) /RMS(Diobs)) for F1 and F2 were 18.9% and 29.9%, respectively  and NOE data showed that the internal structures of F1 and F2 are preserved on DNA complex formation and are similar to those determined previously by NMR spectroscopy  and crys6–2.8 Å) . The Q vectively n. In addectively . For theectively . In the ectively  and 1RNGectively  were useectively  are largectively .NOE contacts, CSP, and TRACT  data for3ODA and 3ODC, respectively)  . In bothctively) . For F1 ctively) A and 2B.To test whether F1 and F2 form a direct F1-F2 interaction in the complex, which could play an important role in determining the mutual orientation of the fingers, sortase-ligated constructs having different labeling in each finger were used to allow selective measurement of NOE contacts between F1 and F2 freed from overlap with other signals . This reThese data were used together to calculate a structure of the complex using a hybrid approach combining information from X-ray crystallography and NMR spectroscopy, using the program XPLOR-NIH . Becausecis modification and AUC experiments. M.F.L. expressed and purified proteins and performed biochemical analysis of full-length PARP-1 and mutants. A.A.R. performed live-cell imaging experiments. S.E., M.F.L., J.M.P., and D.N. wrote the manuscript.D.N. and S.E. designed the project. S.E., W.F.W., and L.E.E. cloned, expressed, and purified proteins for NMR. S.E., W.F.W., and J.C.Y. designed, collected, and processed NMR experiments. S.E., W.F.W., J.C.Y., and D.N. analyzed and assigned NMR data. D.N. and S.E. designed and carried out structural calculations and analyses. S.E. and W.F.W. carried out and analyzed ITC experiments and SAXS experiments. M.F.L. and J.M.P. designed and performed the in


Mycobacterium bovis bacillus Calmette-Guérin (BCG) is known to be only partially effective in inhibiting M. tuberculosis (MTB) multiplication in human. A new recombinant (r) urease-deficient BCG (BCG-dHCM) that secretes protein composed of heat shock protein (HSP)70, MTB-derived CysO and major membrane protein (MMP)-II was produced for the efficient production of interferon gamma (IFN-γ) which is an essential element for mycobacteriocidal action and inhibition of neutrophil accumulation in lungs.Human monocyte-derived dendritic cells (DC) and macrophages were differentiated from human monocytes, infected with BCG and autologous T cells-stimulating activity of different constructs of BCG was assessed. C57BL/6 mice were used to test the effectiveness of BCG for the production of T cells responsive to MTB-derived antigens (Ags).+ T cells through macrophages in a manner dependent on MHC and CD86 molecules. These T cell activations were inhibited by the pre-treatment of Ag-presenting cells (APCs) with chloroquine. The single and primary BCG-dHCM-inoculation produced long lasting T cells responsive to in vitro secondarily stimulation with HSP70, CysO, MMP-II and H37Rv-derived cytosolic protein, and partially inhibited the replication of aerosol-challenged MTB.BCG-dHCM intracellularly secreted HSP70-CysO-MMP-II fusion protein, and activated DC by up-regulating Major Histcompatibility Complex (MHC), CD86 and CD83 molecules and enhanced various cytokines production from DC and macrophages. BCG-dHCM activated naïve T cells of both CD4 and CD8 subsets through DC, and memory type CD4The results indicate that introduction of different type of immunogenic molecules into a urease-deficient rBCG is useful for providing polyclonal T cell activating ability to BCG and for production of T cells responsive to secondary stimulation. Howeverary mice. These o T cells. Among tnfection,12. The nfection,14. Althnfection. The maj T cells,17. The  T cells-20. Ther T cells-20. Actu T cells,21-26 ar+ T cells and naïve CD8+ T cells, and, in mice, produced T cells responsive to H37Rv-derived cytosolic protein[ureC gene of BCG, which encodes urease, from BCG[+ T cells[M. leprae or BCG, and can ligate Toll like receptor (TLR)2 and consequently activate both DC and macrophages[+ T cells and CD8+ T cells[Recently, we have produced recombinant BCG, termed BCG-DHTM which strongly activated human naïve CD4 protein. In the from BCG,20. The  T cells,20. The rophages-31. Also T cells. Since b T cells.Although BCG-DHTM activated both subsets of T cells to produce IFN-γ, production of memory T cells capable of responding to MTB-derived molecules which can be induced chiefly in the activating phase of MTB growth, is needed for the tuberculosis protection. To address this point, we selected CysO (Rv1335 or CFP10A) as the target gene, since CysO also participates in cysteine biosynthesis pathway in MTB. This imIn this study, we introduced CysO gene in combination with HSP70-MMP-II fusion gene into urease-deficient BCG-ΔUT-11-3, and produced new rBCG termed BCG-dHCM, and evaluated its T-cell stimulating activities.+ T cells were removed from either freshly isolated heparinized blood, or cryopreserved PBMCs using immunomagnetic beads coated with anti-CD3 monoclonal antibody (mAb) . The CD3- PBMC fraction was plated on tissue culture plates and the non-plastic adherent cells were removed by extensive washing. The remaining adherent cells were used as monocytes[M. smegmatis. Polyclonal Ab against CysO was produced by immunizing rabbit with the recombinant protein. The rHSP70 protein was purchased  and H37Rv-derived cytosolic protein was produced as described previously[Peripheral blood was obtained from healthy PPD-positive individuals under informed consent. The study was approved by the ethics committee of the National Institute of Infectious Diseases, Tokyo. In Japan, BCG vaccination is compulsory for children (0 ~ 1 year-old). Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus  and cryopreserved in liquid nitrogen until use, as previously described. The viaonocytes. Monocytonocytes,40. Brieonocytes,42. In bonocytes,43, and hsp70 gene were F-Mb70Bal (5′-aaaTGGCCAtggctcgtgcggtcggg-3′) and R-Mb70Eco (5′-aaaGAATTCcttggcctcccggccg-3′). MMP-II sequence from MTB genomic DNA was amplified with primers: F-MMP TB Eco (aattGAATTCatgcaaggtgatcccgatgt) and R-MMP TB Sal (5′-aattGTCGACtcaggtcggtgggcgaga). CysO sequence from MTB genomic DNA was amplified with primers: F-CysO (5′-ggccgggaggccaagaacgtcaccgtatccattcc-3′) and R-CysO: (5′-atcgggatcaccttgcccaccggccacggcgggga-3′). The amplified HSP70 and MMP-II sequence were digested with appropriate restriction enzymes and cloned into parental pMV261H plasmid. For the cloning of CysO sequence into pMV261, In-Fusion HD cloning kit  was used. For replacing kanamycin resistance gene to hygromycin resistance cassette, the Xba I-Nhe I fragment from pYUB854[Spe I-Nhe I fragment of the plasmid[ureC gene was disrupted, was produced as described previously[tnpR)[8 colony forming unit (CFU)/ml at -80°C. Before infection to DC and macrophages, BCGs were counted by colony assay method. There is no significant difference in the in vitro culture growth between BCG-261H and BCG-dHCM.The genomic DNAs were obtained from BCG substrain Tokyo and from MTB H37Rv strain. The oligonucleotide primers used for the amplication of  pYUB854 was clon plasmid. The rBCeviously. The hygy[tnpR). The unm4 live cells were analyzed. For the analysis of the cell surface Ag, the following mAbs were used: FITC-conjugated mAb against HLA-ABC , HLA-DR , CD86 , and CD83 .The expression of cell surface Ag on DC was analyzed using FACSCalibur . Dead cells were eliminated from the analysis by staining with propidium iodide  and 1 × 10+ and CD8+ T cells was conducted by using negative-isolation kits [+ and CD8+ T cells was more than 95% when assessed using FACSCalibur. Naïve CD4+ and CD8+ T cells were produced by further treatment of these T cells with mAb to CD45RO, which were followed by beads coated with goat anti-mouse IgGs Ab . The purity of both subsets of naïve T cells was more than 97%. However, there was no contamination of memory type T cells in the naïve T cell preparations. More than 98% of CD45RA+ T cells was positive in the expression of CCR7 molecule. Memory type T cells were similarly produced by the treatment of cells with mAb to CD45RA Ag. The purified responder cells (1 × 105 per well) were plated in 96-well round-bottom tissue culture plates, and APCs infected with rBCG or pulsed with protein were added to give the indicated APC: T cell ratio. Supernatants of APC-T cell co-cultures were collected on day 4 and the cytokine levels were determined. In some cases, rBCG-infected DC and macrophages were treated with mAb to HLA-ABC , HLA-DR , CD86  or normal mouse IgG. Also, in some cases, immature DC and macrophages were treated with 50 μM of chloroquine (Sigma-Aldrich) for 2 h and subsequently infected with BCG-dHCM. The optimal dose of the Abs and reagents was determined in advance.The ability of DC infected with BCG or pulsed with recombinant protein and BCG-infected macrophages to stimulate T cells was assessed using an autologous APC-T cell co-culture as previously described,45. PuriBiotech). The pur+ and CD8+ T cells, and IL-12p70, IL-12p40, TNFα, IL-1β and GM-CSF produced by DC or macrophages stimulated for 24 or 48 h with rBCGs. The concentrations of these cytokines were quantified using the enzyme assay kits, Opt EIA Human ELISA Set (BD Bioscience).Levels of the following cytokines were measured; IFN-γ produced by CD48 CFU/ml at -80°C. Before the aliquots were used for inoculation, the concentration of viable bacilli was determined by plating on Middlebrook 7H10 agar plate supplemented with Middlebrook OADC enrichment. Three 5-week-old C57BL/6 J mice  per group were inoculated subcutaneously with 0.1 ml of PBS or PBS containing 1 × 103 rBCGs. The animals were kept in specific pathogen free conditions and were supplied with sterilized food and water. Four or 12 weeks after inoculation, the spleens were removed and the splenocytes were suspended at a concentration of 2 × 106 cells per ml in culture medium. The splenocytes were stimulated with an indicated concentration of rMMP-II, rHSP70 (HyTest), rCysO or H37Rv-derived cytosolic protein in triplicates in 96-well round bottom microplates[3 CFU/mouse either BCG-261H or BCG-dHCM for 6 weeks, and were challenged with 100 CFU/lungs of H37Rv by aerosol infection using an automated inhalation exposure apparatus . Six weeks later, bacterial burden in lungs and spleen was assessed by mechanical disruption in PBS with 0.05% v/v Tween 80 and enumerated by colony assay. Animal studies were reviewed and approved by the Animal Research Committee of Experimental Animals of the National Institute of Infectious Diseases, and were conducted according to their guidelines.For inoculation into mice, rBCG and MTB H37Rv strain were cultured in Middlebrook 7H9 medium supplemented with Middlebrook ADC enrichment to log phase and stored at 10roplates,30. The t-test was applied to determine the statistical differences.Student’s M. smegmatis revealed a single band on SDS-PAGE electrophoresis and confirmed by Western blot analysis using polyclonal Ab to CysO (not shown), the immunostimulatory activities of rCysO protein were assessed using rMMP-II of MTB as a positive control. We assessed the activation of APCs from the aspect of phenotypic changes and cytokine production  for 6 weeks were challenged with 100 CFU per lungs of H37Rv by aerosol infection. Six weeks later, the MTB recovered from both lungs and spleen was enumerated (Figure C57BL/6 mice vaccinated with either BCG-261H or BCG-dHCM (1 × 10+ T cells and CD8+ T cells has an essential function in both antimicrobial activity against MTB and limitation of lung inflammation associated with massive accumulation of neutrophils which are recruited by Th17 cells[+ T cells as well as memory type CD8+ T cells capable of producing abundant dose of IFN-γ by responding rapidly to MTB-infected DC and macrophages. From the studies using T cell receptor-transgenic animal model, it is known that it needs several weeks for host T cells to start inhibiting the multiplication of aerosol infected MTB in primary mice[+ T cells. Previous rBCG termed BCG-70M that is BCG introduced with HSP70-MMP-II fusion gene failed to activate memory type T cells through macrophages[+ T cells and naïve CD8+ T cells by BCG-dHCM was dependent on MHC and CD86 molecules expressed on APCs. Thus, BCG-dHCM seems to be stimulating naïve T cells in an Ag-specific fashion. Presumably, APCs infected MTB express various epitopes on their surface, indicating that the presence of clonal diversity of T cells, might provide better control of MTB. Subcutaneous inoculation of BCG-dHCM into C57BL/6 mice produced long lasting T cells responsive to in vitro secondary stimulation. Upon stimulation with not only MMP-II, CysO and HSP70, but also H37Rv-derived cytosolic (Figure M. smegmatis in a LPS-free condition, activated both innate and adaptive cellular responses, since it induced some cytokine production from APCs and phenotypic changes in DC, and also stimulated IFN-γ production from both subsets of T cells. When we introduced CysO gene into urease-deficient rBCG accompanying with HSP70-MMP-II fusion gene, CysO was produced as a part of fusion protein, in fact, the immunogenic function of CysO may be up-regulated because of the chaperone activity of HSP70, that may lead to production of CysO-specific memory T cells in vivo, although we could not elucidate the detailed mechanisms of the immunological function of CysO. Actually, mice inoculated with BCG-dHCM produced T cells that respond to CysO vigorously. These results indicate that CysO protein was certainly secreted from rBCG in APC and was used for T cell activation. CysO expression in MTB is inhibited in hypoxic condition and is induced by reaeration[IFN-γ produced by activated type 1 CD417 cells,46. Therary mice,6. It isary mice. Howeverary mice,17. Thusary mice, and alsary mice-31, MMP-rophages, thus, trophages,31. In taeration, thus, Caeration, and by aeration, after Min vivo, and may provide one possible tool for the development of better vaccine against tuberculosis.These results indicate that the secretion of polycomponent antigenic molecules can efficiently produce polyclonal Ag-specific T cells responsive to secondary stimulation Mycobacterium bovis BCG; M: Mycobacterium; r: Recombinant; MTB: M. tuberculosis; HSP: Heat shock protein; MMP: Major membrane protein; IFN-γ: Interferon-gamma; DC: Dendritic cells; Ag: Antigen; MHC: Major histcompatibility complex; APC: Antigen-presenting cell; TLR: Toll-like receptor; PBMC: Peripheral blood mononuclear cell; mAb: Monoclonal antibody; MOI: Multiplicity of infection; CFU: Colony forming unit.BCG: The authors declare that they have no competing interests.YT and MM participated in the design of the study and carried out the cell culture experiments, YM and TT carried out animal studies, YT and TM conducted the construction of recombinant BCG. YT, YM and MM were involved in the preparation of the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/179/prepub


With the development of new technologies in transcriptome and epigenetics, RNAs have been identified to play more and more important roles in life processes. Consequently, various methods have been proposed to assess the biological functions of RNAs and thus classify them functionally, among which comparative study of RNA structures is perhaps the most important one. To measure the structural similarity of RNAs and classify them, we propose a novel three dimensional (3D) graphical representation of RNA secondary structure, in which an RNA secondary structure is first transformed into a characteristic sequence based on chemical property of nucleic acids; a dynamic 3D graph is then constructed for the characteristic sequence; and lastly a numerical characterization of the 3D graph is used to represent the RNA secondary structure. We tested our algorithm on three datasets: (1) Dataset I consisting of nine RNA secondary structures of viruses, (2) Dataset II consisting of complex RNA secondary structures including pseudo-knots, and (3) Dataset III consisting of 18 non-coding RNA families. We also compare our method with other nine existing methods using Dataset II and III. The results demonstrate that our method is better than other methods in similarity measurement and classification of RNA secondary structures. As a bridge of information transmission, RNAs carry a wide variety of functions in biological systems, including performing catalytic function, regulating gene expression, and carrying genetic information . In liviRecently, ncRNAs have become a focus for both computational and experimental researches . Though It is known that the functions of ncRNAs are mostly determined by their structures . Though Historically, dynamic programming based algorithms with various scoring functions have been widely used to measure the similarities among RNA secondary structures –19. For In order to measure RNA similarity more efficiently, various alternative techniques have been tested. For example, a novel 2D graphical representation of RNA secondary structure was proposed in . HoweverIn this paper, we proposed a novel dynamic 3D graphical representation of RNA secondary structures based on their sequences, chemical properties, and structural information. We evaluated our algorithm on three sets of RNA secondary structures including 9 viral RNAs, 33 RNAs with complex secondary structures, and 120 non-coding RNAs. A comparison study showed that our novel method outperformed other nine methods in deciphering RNA similarity.We adopted three RNA datasets in this study: (1) Dataset I consisting of 3'-terminus of RNAs from nine viruses reported by Bol see  28];  amino M = {A,C} and keto K = {G,U}, (ii) purine R = {A,G} and pyrimidine Y = {C,U}, and (iii) weak H-bonds W = {A,U} and strong H-bonds S = {C,G}. According to the base classification scheme (i), (ii), and (iii), a characteristic sequence can be represented by three 3D graphs through three maps vely see , where AG = g1g2g3…gn be the characteristic sequence of an RNA secondary structure, where n is the length of G. Each base gi can be mapped into three dots φ1(gi) = , φ2(gi) = , and φ3(gi) =  where i = 1, 2, …, n. By connecting dots in φ1(gi) =   in order, we can obtain an M-K curve to represent the characteristic sequence G. Similarly, by connecting dots in φ2(gi)  and φ3(gi)  respectively, we can obtain R-Y and W-S curves. Taking the secondary structure of TSV-3 as an example, we plotted its M-K, R-Y, and W-S curves in Specifically, let In this section, we introduced 3 properties showing 3 theoretical advantages of our method including non-degenerative (no information loss), easily reflecting the content of stem and loop, and the distribution of base frequencies. The proof of the properties were provided in The mapping between an RNA secondary structure and its dynamic 3D graphical representation is one to one, and the mapping on the X-Y plane is non-degenerative. Thus no information is lost.Our dynamic 3D graphical representation can easily reflect the content of bases and proportion of stem and loop structures.This property shows that a few information on the base distribution and compositions of RNA secondary structure, such as the proportion of stem and loop structures, can be intuitively reflected by the dynamic 3D graphical representation. It is of note that the proportion of stem and loop structures is extremely important for RNA secondary structure prediction .A′, G′, C′, U′, A, G, C, U be a′, g′, c′, u′, a, g, c, u, and i = 1, 2, …, n, where n is the length of the characteristic sequence, then For a characteristic sequence of the RNA secondary structure, let the frequencies of bases xi1, yi1, zi1), , and , i = 1, 2, …, n, where n is the length of the structure. We draw the geometrical center of the three curves to construct a 36-dimensional vector denoted by For an RNA secondary structure, we have three sets of points  the Euclidean distance between their end-points (in graph) and (2) the cosine of the angle between the two vectors. Clearly, the smaller is the quotient, the more similar are the two RNA secondary structures.Dataset I, and listed their pairwise similarities in We drew in As Dataset II, whose characteristic sequences were shown in To further evaluate the sensitivity of this algorithm in measuring the similarities among complex RNA secondary structures, we applied it to Based on , 38, theDataset II.We plotted the similarities among the 33 RNA secondary structures (based on their 36D vector representations) in Dataset III, whose characteristic sequences were shown in By a similar process, we constructed a phylogenetic tree  for the Dataset II and III, and the RNA similarities measured by these methods were shown as UPGMA trees in We compared our similarity measure with other nine popular RNA comparison methods, including a similarity metric based on the wavelet decomposition of the TV-Curve of ncRNA , LZ compDataset II. In addition, except for the method provided by Luo et al. [Dataset III. Specifically, the method by Liu et al. [Dataset II  17 complicated RNA secondary structures from RNase P database: Klebsiella pneumoniae, Serratia marcescens, Escherichia coli K-12 W3110, Chromatium vinosum, Chlorobium limicola thiosulfatophilum, Chlorobium tepidum, Bacillus subtilis 168, Enterococcus (ex-Streptococcus) faecalis, Calothrix PCC7601, Anabaena PCC7120, Synechocystis PCC6803, Thermococcus celer AL-1, Pyrococcus horikoshii strain OT3, T. Litoralis, Pan troglodytes, Macaca mulatta, Pongo pygmaeus. (B) 16 RNA secondary structures with pseudo-knots from Pseud Base: PKB44, PKB46, PKB4, PKB42, PKB43, PKB94, PKB114, PKB84, PKB134, PKB135, PKB131, PKB132, PKB144, PKB140, PKB142, PKB143.(DOC)Click here for additional data file.S2 Fig(A) 5S rRNA (downloaded from Gutell Lab CRW Site in RNAstrand database and belonging to the RF00001 family of Rfam database). (B) Gammaretro_CES (RF00374). (C) Hepatitis delta virus ribozyme (RF00094). (D) Vimentin3 (RF00109). (E) Corona_pk3 (RF00165). (F) Y_RNA (RF00019). (G) s2m (RF00164). (H) Hammerhead ribozyme (type III) (RF00008). (I) Ciliate telomerase RNA (RF00025). (J) R2 RNA element (RF00524). (K) Hammerhead ribozyme (type I) (RF00163). (L) Vertebrate telomerase RNA (RF00024). (M) rne5 (RF00040). (N) RNase MRP (RF00030). (O) 7SK RNA (RF00100). (P) RNAIII (RF00503). (Q) RydC RNA (RF00505). (R) Bicoid 3 prime-UTR regulatory element (RF00551) (non-coding RNA secondary structures belonging to B-R are obtained from Rfam database in RNAstrand database).(DOC)Click here for additional data file.S3 Fig(DOC)Click here for additional data file.S4 Fig(DOC)Click here for additional data file.S5 Fig(A) The phylogenetic tree based on the Euclidean distance. (B) The phylogenetic tree based on the Angle.(DOC)Click here for additional data file.S6 Fig(A) The phylogenetic tree based on the Euclidean. (B) The phylogenetic tree based on the Angle.(DOC)Click here for additional data file.S7 Fig(A) The phylogenetic tree based on the Euclidean distance. (B) The phylogenetic tree based on the Angle.(DOC)Click here for additional data file.S8 Fig(A) The phylogenetic tree based on the Euclidean distance. (B) The phylogenetic tree based on the Angle.(DOC)Click here for additional data file.S9 Fig(DOC)Click here for additional data file.S10 Fig(DOC)Click here for additional data file.S11 Fig(A) The phylogenetic tree based on the Euclidean distance. (B) The phylogenetic tree based on the Angle.(DOC)Click here for additional data file.S12 Fig(A) The phylogenetic tree based on the Euclidean distance. (B) The phylogenetic tree based on the Angle.(DOC)Click here for additional data file.S13 Fig(DOC)Click here for additional data file.S14 Fig(DOC)Click here for additional data file.S15 Fig(DOC)Click here for additional data file.S16 Fig(DOC)Click here for additional data file.S17 Fig(A) The phylogenetic tree based on the Euclidean distance. (B) The phylogenetic tree based on the Angle.(DOC)Click here for additional data file.S18 Fig(A) The phylogenetic tree based on the Euclidean distance. (B) The phylogenetic tree based on the Angle.(DOC)Click here for additional data file.S19 FigA(A′) sequences. (B) The phylogenetic tree based on the non-C(C′) sequences. (C) The phylogenetic tree based on the non-G(G′) sequences.(A) The phylogenetic tree based on non-(DOC)Click here for additional data file.S20 FigA(A′) sequences. (B) The phylogenetic tree based on the non-C(C′) sequences. (C) The phylogenetic tree based on the non-G(G′) sequences.(A) The phylogenetic tree based on the non-(DOC)Click here for additional data file.S1 Software(ZIP)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.  •Crystal structures reveal how CGRP and AM peptides bind their heterodimeric receptors•CGRP and AM occupy a shared binding site on CLR with minimal contact to the RAMPs•Peptide binding modes were confirmed for intact receptors in cells by mutagenesis•Peptide selectivity arises from RAMP-specific contacts and subtle alteration of CLR Booe et al. report two crystal structures that reveal how selectivity of the GPCR CLR for CGRP and AM peptides is modulated by RAMP proteins. The peptides similarly occupy a shared binding site on CLR. RAMPs augment the binding site with distinct contacts to the peptides and subtly alter CLR. G protein-coupled receptors (GPCRs) are a large family of cell surface receptors that regulate a multitude of biological processes in response to a diverse array of stimuli and they are important drug targets. The class B/Secretin family GPCRs in humans include 15 receptors that are activated by diverse neuropeptides, peptide paracrine factors, and peptide endocrine hormones . These rAlthough this model of binding is valid for several class B GPCRs, it cannot apply to all class B receptors. In particular, there are problems understanding the binding of members of the calcitonin (CT) family of peptides; calcitonin gene-related peptides alpha and beta , adrenomedullin (AM), adrenomedullin 2/intermedin (AM2), amylin (Amy), and CT . These C1 receptor, and CLR:RAMP3 binds both AM and AM2 with high affinities and is called the AM2 receptor. Amy by itself has a low affinity for the class B CT receptor (CTR); however, when CTR associates with any of the RAMPs, its affinity for Amy is markedly increased , is dependent on association of CLR with one of three accessory membrane proteins that determine ligand selectivity; receptor activity-modifying proteins (RAMPs) 1, 2, or 3 . RAMPs hncreased . CTR aloncreased . RAMPs pCrystal structures are available for ligand-free and small molecule antagonist-bound CLR:RAMP1 and ligand-free CLR:RAMP2 ECD complexes, but these provide little insight into how peptides bind or how RAMPs determine selectivity . Extensi5 linker were designed to ensure complex stability and enforce 1:1 CLR:RAMP stoichiometry. The tethered RAMP1-CLR ECD fusion was a monomer, whereas the tethered RAMP2-CLR ECD fusion purified as a dimer, but the physiological relevance of oligomerization is unknown. Both proteins selectively bound their respective peptides but failed to yield crystals in the presence of peptides.We previously reported a tethered fusion protein approach to engineer the CLR:RAMP1 and CLR:RAMP2 ECD complexes for crystallization , inspire1 receptor ECD complex hindered the crystallization efforts. We produced new constructs with a (Gly-Ser-Ala)3 tether designed to decrease flexibility and we identified a single amino acid substitution in the RAMP2 ECD, L106R, which prevented dimerization of the tethered RAMP2-CLR fusion protein  (1 receptor with RAMP2 [L106R] exhibited wild-type response to AM (3-CLR ECD grown in the presence of AM(25-52)NH2 were readily obtained (3-CLR ECD grown in the presence of CGRP(20-37)NH2 diffracted poorly (data not shown); fortunately, high-quality crystals were obtained in the presence of a high-affinity CGRP analog CGRP(27-37)NH2  (50 ∼0.46 μM) than CGRP(8-37)NH2 (IC50 ∼2 μM)  G. The CG than AM H. The cr than AM I. The CGRwork and Rfree values (1 receptor fusion were present in the asymmetric units. Molecule A (Mol A) of the CGRPmut-bound structure had the best electron density and lowest B-factors  and only 6% is from RAMP1 (29 Å2). More ASA is buried at the interface with AM, but the majority is still from CLR, 90% (781 Å2), whereas RAMP2 contributes only 10% (85 Å2).CGRPmut and AM primarily contact CLR, but key RAMP contacts are also formed  and S2. receptor C and 2D.receptor B and 2D.receptor A and 2B,receptor C and 2D.receptor A and 2B.receptor C and 2D.receptor D. For thSuperpositions of CGRPmut-bound and ligand-free CLR:RAMP1 complexes  revealedThe CGRPmut-bound structure explains antagonism by the CGRP receptor-selective small molecule drugs olcegepant and telcagepant. Superposition of the CGRPmut-bound and drug-bound structures  indicateSuperposition of the AM-bound and ligand-free CLR:RAMP2 complexes  revealedTo validate the structures, we constructed several Ala substitution mutants in the CLR and RAMP2 ECDs, and they were analyzed for their effects on peptide-stimulated cAMP formation in COS-7 cells. For the CGRP receptor, Ala substitution of CLR W69, D70, K103, or Y91 significantly reduced CGRP potency, likely due to the structural roles of these residues . Ala sub1 receptor, Ala substitution of CLR W72, F92, F95, W121, or Y124, which are contacted by AM in the structure, resulted in >40-fold decreases in AM potency  and we tested their response to CGRP and AM in the cAMP assay C and 6D.2 [F37Y] retained the ability to bind the CGRP receptor ECD complex , but how this region interacted with the receptor was unclear . Prior t1 receptor, and truncation beyond residue 38 diminished binding even though the K38-V41 side chains were not important NH2 significantly diminished its binding. The lack of Glu at RAMP1 position 74 would disfavor strong AM binding. RAMP1 W84 enables strong CGRP binding by contacting F37, but this contact alone is apparently insufficient for selectivity because AM(37-52)NH2 [Y52F] did not gain affinity for the CGRP receptor ECD complex.Distinct RAMP binding site augmentation clearly contributes to peptide selectivity . RAMP2 E2 receptor ECD complex NH2 and crystallized with a reservoir solution of 22% PEG3350, 8% Tacsimate (pH 6.0) for the CGRP receptor complex or 19% PEG3350, 0.1 M Tris-HCl (pH 8.3), 225 mM sodium acetate, and 20% ethylene glycol for the AM1 receptor complex. Diffraction data collected at the APS synchrotron were processed with HKL2000 (The tethered MBP-RAMP1 ECD-CLR ECD and MBP-RAMP2 ECD [L106R]-CLR ECD proteins were complexed with CGRP(27-37)NH HKL2000  and the  HKL2000 . The str HKL2000 , rebuilt HKL2000 , and ref HKL2000 . DetailsTransfection of COS-7 cells, cAMP assay, ELISA for cell surface expression, and data analysis were as previously described .1 receptors; J.B., G.K., J.S., R.M.B., and D.R.P. designed and constructed CLR mutants and performed and analyzed cell-based assays for the CGRP receptor; J.S. performed RAMP3 homology modeling; M.L.W. performed peptide binding experiments; P.W.H. and M.A.B. synthesized CGRP analog; A.A.P. designed and managed the structural project, solved/refined the structures, and wrote the manuscript with D.R.P. and D.L.H.J.M.B. produced and characterized proteins, performed crystallization, collected diffraction data, and constructed CLR and RAMP mutants; C.S.W., M.A.J., and D.L.H. designed, performed, and analyzed cell-based assays for the CGRP and AM


Pseudomonas putida, various strategies for the over-expression of P14K are needed to facilitate further application of NHase.The activator P14K is necessary for the activation of nitrile hydratase (NHase). However, it is hard to be expressed heterogeneously. Although an N-terminal strep tagged P14K could be successfully expressed from Bordetella petrii DSM 12804. The stability of gst-P14K was demonstrated to be higher than that of the his-P14K. In addition, the Ser115 in the characteristic motif CXLC-Ser115-C of the active center of NHase was found to be unnecessary for NHase maturation.P14K was successfully expressed through fusing a his tag (his-P14K), and was over-expressed through fusing a gst tag (gst-P14K) at its N-terminus in the NHase of Our results are not only useful for the NHase activator expression and the understanding of the role of Ser115 during NHase activation, but also helpful for other proteins with difficulty in heterologous expression. Nitrile hydratase (NHase) is a multi-subunits industrial enzyme, which can efficiently catalyze the nitrile to the corresponding amide. According to the different metal ions in the active center, the NHase could be classified into the two types: the iron type NHase (Fe-NHase)  and the Bordetella petrii DSM 12804  and those in Bacillus pallidus RAPc8  [Rhodococcus rhodochrous J1 [Rhodococcus jostii RHA1 [Pseudomonas putida NRRL-18668 [Pseudomonas chlororaphis B23 [Rhodococcus sp. N-774 [B. pallidus RAPc8, Comamonas testosteroni 5-MGAM-4D and P. putida NRRL-18668 [P. putida was successfully expressed through fusing a strep tag at its N-terminal, then a novel NHase maturation mechanism, self-subunit swapping, was discovered [NHase is composed of α- and β-subunits . The moltii RHA1  and PseuRL-18668  as well phis B23  and RhodRL-18668 , 13. UntAdditionally, all the α-subunits of NHases share a conserved characteristic motif CXLC-Ser-C in the active center , 14, 15,B. petrii DSM 12804 was researched. The activator P14K was successfully expressed through fusing a his tag, and its expression and stability were further enhanced by fusing a gst tag at its N-terminus. In addition, we found that the Ser115 in the characteristic motif CXLC-Ser-C of the active center of NHase from B. petrii is not necessary for the NHase maturation. These results are useful for the expression of NHase activator and the understanding of the role of Ser115 during the NHase maturation. Further, the strategy for enhancing the stability of P14K is very beneficial for other proteins with difficulty in expression.In this study, the NHase from ABP) were obtained from B. petrii DSM 12804, in which A gene encodes α-subunit, B gene encodes β-subunit and P gene encodes P14K  was constructed and transformed into E. coli for NHase expression. The calculated molecular mass of recombinant NHase was in accordance with that of the bands by SDS-PAGE analysis  with that in P. putida NRRL-18668, it is proposed that the P14K in B. petrii is also necessary for NHase activation. Here, the NHase expressed by pET-ABP plasmid exhibited enough activity (more than 10 U/mg), indicating the successful expression of NHase and P14K, though the P14K band was not detected in SDS-PAGE  were changed to the GSS sequence of the designed his-P14K, the his-P14K would be more stable in terms of the N-end rule. The plasmid pET-AB(his-P) including NHase encoding genes A (α-subunit), B (β-subunit) and his-P (his-tagged P14K) was transformed into E. coli and was applied for NHase expression , a plasmid including NHase encoding genes A (α-subunit), B (β-subunit) and P-his , were applied for NHase expression. As a result, the enzymatic activity detected was comparable to that of the original NHase, however, P14K-his was barely detected in SDS-PAGE , a plasmid including NHase encoding genes A (α-subunit), B (β-subunit) and gst-P (gst-tagged P14K), were applied for NHase expression. As shown in Fig. P. putida plays an important role in NHase maturation [The gst tag 26 kDa) encodes a highly soluble protein, which can help other proteins rapidly fold into a stable protein [ kDa encoturation  and the Moreover, the protein stability of his-P14K and gst-P14K in the cell-free extracts was compared during storage at room temperature by SDS-PAGE analysis. As shown in Fig. 2   was more than that of the direct transfer (type II) , which indicated the type II pathway without the involvement of the Ser115 seemed easier to take place. Thus, a mutant NHase with the Ser115 mutated to Ala115 was designed to investigate the necessity of the Ser115, considering the structural similarity of the serine and alanine except for the -OH. The transformed cells carrying pET-(A*)BP, a plasmid including NHase encoding genes A* (mutant α-subunit with Ser115 to Ala115), B (β-subunit) and P (P14K), were applied for the mutant NHase expression  were isolated from B. petrii DSM 12804 . The plasmid pET-24a(+) carrying the respective genes ABP, AB(his-P), AB(P-his), AB(gst-P) and (A*)BP was transformed into E. coli BL21 (DE3) cells (host strain) for heterologous expression.NHase genes (ABP of B. petrii were cloned through the polymerase chain reaction (PCR) with the primers A-up (NdeI recognition sites) and P-down (EcoRI recognition sites) (Table ABP and the vector pET-24a(+) were treated by the NdeI and EcoRI, and the resultant ABP was then inserted into the pET-24a(+) for the pET-ABP construction. The pET-AB(P-his) construction was similar to pET-ABP with the primer pairs A-up and P-down(his) (EcoRI recognition sites). An overlap extension PCR was conducted to produce plasmid pET-AB(his-P) through two rounds of PCR. The first round of PCR was performed using the primer pairs A-up and B-down(his) and P-up(his) and P-down, respectively, the plasmid pET-ABP was used as the template. The second round of PCR was performed to produce the full-length AB(his-P), using the primers A-up and P-down and mixing equal molar amounts of the first-round products as template. The pET-AB(gst-P) construction was similar to pET-AB(his-P) with the primer pairs A-up and B-down(gst), and P-up(gst) and P-down (the gst-P gene synthesized by Sangon Biotech Ltd.). The plasmid pET-(A*)BP construction was conducted with the primers A*-up and A*-down using pET-ABP as the template, followed by DpnI digestion to degrade the template plasmid and then transformed into E. coli BL21 (DE3).The NHase genes s) Table . The PCRE. coli cells carried the corresponding recombinant plasmids were cultivated in the TB medium to 0.8 of A600 with kanamycin (37 °C), and then 0.4 mM isopropyl β-D-thiogalactopyranoside (IPTG) was added. The transformed cells were then cultivated at 24 °C for 16 h.The recombinant g for 10 min. Protein was preliminarily purified by the ammonium sulfate fractionation (30–70 %) followed by dialysis against KPB. The dialyzed protein ingredients of NHase were purified using a DEAE-Sephacel column (3 × 5 mL)  equilibrated with KPB. The fraction containing target proteins was gradually eluted off by a linear concentration of KCl (0–0.5 M) during purification and then the collected proteins were pooled and followed by ammonium sulfate fractionation (70 %). After centrifugation, the dissolved precipitate (KPB containing 0.2 M KCl) was applied to a HiLoad 16/60 Superdex 200 prep grade column  equilibrated with 0.2 M KCl-containing KPB. The enzymes were then eluted from the HiLoad 16/60 Superdex 200 prep grade column. The fractions containing the enzymes during the purification steps were revealed by SDS-PAGE.The purifier AKTA instrument  was used for proteins purification. All the purifications were kept in the potassium phosphate buffer  at 4 °C. The cell extracts were centrifuged at 18000 × The NHase activity was measured in a reaction system (500 μL) consisted of NHase (0.1 μg), the substrate (20 mM 3-cyanopyridine) and the buffer , which was placed for 20 min at 20 °C and stopped by addition of 500 μL acetonitrile. The product concentration (nicotinamide) was detected with HPLC (high-pressure liquid chromatography) to measure the NHase activity . The amohttp://services.mbi.ucla.edu/SAVES/) were applied for the prediction of protein structure and stereochemical analysis. The final models that displayed good geometry  were used in this study.The Modeller 9.7  and the http://ncbr.chemi.muni.cz/triton/) consisting of three modules of MOPAC, MODELLER and DRIVER [TRITON was used for modeling enzymatic reaction, which is freely supported by the National Center of Biomolecular Research (d DRIVER . The optd DRIVER .