Published data regarding the associations between genetic variants and asthma risk in Chinese population were inconclusive. The aim of this study was to investigate asthma susceptible genes in Chinese population.The authors conducted 18 meta-analyzes for 18 polymorphisms in 13 genes from eighty-two publications.A Disintegrin and Metalloprotease 33 (ADAM33) T1-C/T , Angiotensin-Converting Enzyme (ACE) D/I , High-affinity IgE receptor β chain (FcεRIβ) -6843G/A , Interleukin 13(IL-13) -1923C/T , IL-13 -2044A/G , Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES) -28C/G , Tumor Necrosis Factor-α (TNF-α) -308G/A. After subgroup analysis by age, the ACE D/I, β2-Adrenergic Receptor (β2-AR) -79G/C, TNF-α -308G/A, Interleukin 4 receptor(IL-4R) -1902G/A and IL-13 -1923C/T polymorphisms were found significantly associated with asthma risk in Chinese children. In addition, the ACE D/I, FcεRIβ -6843G/A, TNF-α -308G/A, IL-13 -1923C/T and IL-13 -2044A/G polymorphisms were associated with asthma risk in Chinese adults.Seven polymorphisms were found being associated with risk of asthma, namely: ADAM33, FcεRIβ, RANTES, TNF-α, ACE, β2-AR, IL-4R and IL-13 genes could be proposed as asthma susceptible genes in Chinese population. Given the limited number of studies, more data are required to validate these associations. Asthma is one of the most common chronic respiratory diseases, affecting about 300 millions of children and adults worldwide. In ChinIt is well accepted that asthma is a complex disease and both genetic and environmental factors contribute to its inception and evolution,5. Many In recent years, host genetic susceptibility to asthma has been a research focus in scientific community in China. Many genes were suggested as asthma risk factors for Chinese population; however, many of the studies drew incompatible or even contradictory results. Considering a small number of sample size may be lack of power to reveal the reliable conclusion, we carried out a meta-analysis to assess the susceptible genes for asthma in Chinese population. To our knowledge, this is the first comprehensive and largest genetic meta-analysis conducted in people of Chinese descent for any respiratory diseases.asthma or asthmatic', in combination with 'polymorphism or variant or mutation' and 'Chinese or China' for Medline (Ovid), Pubmed, Embase, ScienceDirect, Springer database; 'asthma or asthmatic', in combination with 'polymorphism or variant or mutation' for CNKI database, Wanfang database, Weipu database and CBM database. All languages were included. The following criteria were used for selecting literatures in the meta-analysis: (1) the study should evaluate the association between genetic variants and risk of asthma in Chinese population from either mainland, overseas or both, (2) the study should be a case-control design published in a journal (3) genotype distributions in both cases and controls were available for estimating an odds ratio with 95% confidence interval (CI) and P value, (4)genotype distributions of control population must be consistent with Hardy-Weinberg equilibrium(HWE), P > 0.05 (5) the polymorphism for data synthesis should be studied in at least three case-control studies, (6) polymorphisms for data synthesis should be characterized as -A/B, with the following genotypes: AA, AB and BB. Accordingly, the following exclusion criteria were used: (1) abstracts and reviews, (2)genotype frequency not reported, (3) repeated or overlapping publications (4) polymorphisms with data less than three case-control studies (5) genotype distributions of control population not consistent with HWE, (6)genetic variants not characterized as -A/B. For duplication or overlapping publications, the studies with larger number of cases and controls or been published latest were included.We conducted a literature search by using the electronic database Medline (Ovid), Pubmed, Embase, ScienceDirect, Springer, CNKI, Wanfang database, Weipu database and CBM database to identify articles that evaluated the association between genetic variants and the risk of asthma in Chinese population . The search terms were used as follows: 'Two independent authors (Xiaobo Li and Yonggang Zhang) checked all potentially relevant studies and reached a consensus on all items. In case of disagreement, a third author(Jie Zhang) would assess these articles. The following data were collected from each study: first author, year of publication, location of the people, ages, genotype frequencies in cases and controls.2 Xtest http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl.For each case-control study, we first examined whether the genotype distribution in control group was according to Hardy-Weinberg equilibrium by Pearson's 2X-based Q statistic and was considered statistical significant at P value < 0.10. 2 Iwas used to measure the percentage of variability in point estimated that due to heterogeneity rather than sampling error. When the P-value is > 0.10, the pooled OR was calculated by the fixed-effects model, otherwise, a random-effects model was used. To evaluate the age-specific effects, subgroup analyses were performed by age for polymorphisms which were investigated in a sufficient number of studies(data were available from at least three case-control studies for at least one subgroup). Publication bias was examined by using the funnel plots, Begg's test and Egger's test[Any polymorphism that had been studied in at least three case-control studies was included in the meta-analysis. The strength of the associations between asthma risk and genetic variants were estimated by ORs and 95% CIs. The statistical significance of summary ORs were assessed by Z-test. The evaluated genetic models for each study were based mostly on those used in primary studies. Heterogeneity was evaluated by a er's test. The funThe selection process is shown in Figure 2X-based Q statistic and was considered statistical significant at P-value <0.10. When the P-value is less than 0.10, the pooled OR of each meta-analysis was calculated by the fixed-effects model; otherwise, a random-effects model was used. The chosen models to synthesize the data for each polymorphism can be seen in Table For each polymorphism, heterogeneity was analyzed by a ADAM33 T1-C/T , ACE D/I, FcεRIβ -6843G/A , IL-13 -1923C/T, IL-13 -2044A/G, RANTES -28C/G , TNF-α -308G/A . These results indicated that these polymorphisms were significant associated with asthma risk in Chinese population. All results for all 18 meta-analyzes are summarized in table Forest plots of each polymorphism can be seen in Figure ACE D/I, β2-AR -79G/C, TNF-α -308G/A, IL-4R -1902G/A and IL-13 -1923C/T) in children and five polymorphisms in adults.To evaluate the age-specific effects, subgroup analyses were performed by age for polymorphisms which were investigated in a sufficient number of studies(data were available from at least three case-control studies for at least one subgroup). Three subgroups were used: adults, children, others(ages in these case-control studies were not mentioned or mixed with adults and children). Briefly, we obtained significant results from five polymorphisms were statistically associated with increased risk of asthma. In order to analysis the age-specific associations, subgroup analysis were performed by age. The ACE D/I, β2-AR -79G/C, TNF-α -308G/A, IL-4R -1902G/A and IL-13 -1923C/T polymorphisms were found being associated with asthma risk in Chinese children, while the ACE D/I, FcεRIβ -6843G/A, TNF-α -308G/A, IL-13 -1923C/T, IL-13 -2044A/G polymorphisms were associated with asthma risk in Chinese adults. Given that the data for each polymorphism were from at least three case-control studies, the obtained results could be more precise than results obtained form any individual study.The aim of meta-analysis is to combine results from studies on the same topic and to produce more precise results. The current study is to reveal the roles of genetic variants and their associations with risk of asthma in Chinese population. In summary, we finally identified 18 polymorphisms in 13 genes. Among them, seven polymorphisms  in Chinese population. For A vs G, the pooled OR is 1.26 with 95%CI: 1.08-1.47 in this study, which suggested a weaker association between this polymorphism and asthma risk in Chinese population.The TNF-α gene, encodes a key proinflammatory cytokine in airway, is located on an asthma susceptible region-chromosome 6p. The TNF-α protein plays a central role in inflammation and involves in pathogenesis of asthma. Several polymorphisms have been identified in this gene, such as -308A/G, -238A/G. The -308A/G polymorphism in the promoter may affect the expression of this cytokine, which may affect the occurrence of asthma. In the meta-analysis performed by Gao and colleagues, they foIL-4 gene is located on chromosome 5q31, it was suggested to be associated with asthma risk, including elevated serum IgE levels and airway hypersensitiveness. A few studies indicated the -589C/T polymorphism in the promoter as a risk factor for asthma, but with inconclusive results. Li and colleagues performed a meta-analysis and found the T allele was associated with decrease risk of asthma[.78-0.94). HoweverIL-4 receptor gene is located on chromosome 16 p12.1-p11.2. Some polymorphisms had been identified as risk factors for asthma, such as -1902G/A and -223G/A. Our results indicated the -1902G/A polymorphism was associated with increased risk of asthma in Chinese children, but not in Chinese adults. The results also indicated the -223G/A polymorphism was not associated with risk of asthma in Chinese population.IL-4 and IL-13 signal through binding to a receptor complex comprised of the IL-13Rα1 and IL-4Rα with subsequent phosphorylation of JAKs and STAT6. IL-4 reFcεRIβ gene is a major candidate gene, involving in the pathogenesis of asthma. It is located on the chromosome 11q13. The -6843G/A polymorphism, leading change in an amino acid sequence at residue 237 from glutamic acid to glycine, is associated with increased IgE levels in atopic asthmatic children. In Chinese population, the -6843G/A polymorphism is the most extensively studied polymorphism in FcεRIβ gene. Our study revealed this polymorphism as a risk factor of asthma in Chinese population. Chinese who carry the GG or GA genotype have an 49% increased risk of asthma than AA carriers. Our results also demonstrated the -109C/T polymorphism in this gene was not associated with increased risk of asthma in Chinese population.The ADAM33 T1-C/T, ACE D/I, IL-13 -1923C/T, RANTES -28C/G and IL-13 -2044A/G polymorphisms were associated with risk of asthma in Chinese population by using meta-analyzes. Some results are similar to other studies performed in other ethnic- groups and some are not. In future, more published results should be included to update and validate these associations in Chinese population.Up to date, we first found that ADAM33 T1-C/T, ACE D/I, IL-13 -1923C/T, RANTES -28C/G, as risk factors for asthma was greater than that of FcεRIβ -6843G/A, IL-13 -2044A/G and TNF-α -308G/A.In this study, the rigorous inclusive criteria made the results more precise. Any study in which genotype distribution of control group divorced from HWE was excluded. In this meta-analysis, 11 polymorphisms were synthesized by using the fixed-effect model, 7 used random-effects model. Because the fixed-effect model is more precise than random effect model, the strength of evidence of FcεRIβ -6843G/A. After subgroup analyses by age, the heterogeneity was effectively decreased or removed in adults. Possible explanation may be that differences in etiology may exist in difference age groups. Another important factor contributing to heterogeneity was that homogeneity in either the case and control groups was uncertain. Ideally, all cases and controls in this meta-analysis should be matched for age, sex, atopic status and environmental exposures. However, these issues could not all be explained precisely because of insufficient clinical information for individual person. In addition, because this study is based on population of Chinese descent with the same genetic background, so the similarity of these studies might be very good, despite most studies were conducted in different areas of China.The heterogeneity of clinical information among studies should also be mentioned. Heterogeneity is an important issue when interpreting the results of meta-analysis. Significant heterogeneity existed in overall comparisons in a few meta-analyses, such as P values of Begg's test were more than 0.05 in 18 meta-analyses, we could not rule out this possibility, because for some polymorphisms, the included number of studies were relatively small. Third, this study didn't included some polymorphisms with lack of number of studies, or polymorphisms which were not characterized as -A/B for lack of quality analysis for HWE, some polymorphism, such as GSTM1-P/N, or HLA DR1 alleles and MHC alleles were not included, future studies should performed to analysis the effect of these polymorphism in Chinese population.Some limitations of this meta-analysis should be acknowledged when explaining our results. First, only published articles in the selected electronic databases were included in this study, it may be possible that some studies were not included in those databases or some unpublished studies which had null results, which might bias the results. Second, due to lack of sufficient data, the homogeneity in either the case and control groups was uncertain and data were not stratified by other factors such as atopic status or sex. The tests for gene-environment interactions were not carried out either. Third, publication bias may affect the results. Although ADAM33 gene, the D/I polymorphism in ACE gene, the -6843G/A polymorphism in FcεRIβ gene, the -1923C/T polymorphism in IL-13 gene, the -2044A/G polymorphism in IL-13 gene, the -28C/G polymorphism in RANTES gene and the -308G/A polymorphism in TNF-α gene are associated with asthma risk in Chinese population. And these results may also implicate in personalized therapy for asthma in Chinese population. In future, more studies should be conducted to investigate the gene-gene and gene-environment interactions between these polymorphisms in Chinese population.To our knowledge, this is the first and most comprehensive genetic meta-analysis to date conducted in Chinese descent for any respiratory diseases. In conclusion, this meta-analysis indicated the T1-C/T polymorphism in ADAM33: A Disintegrin and Metalloprotease 33; FcεRIβ: High-affinity IgE receptor β chain; ACE: Angiotensin-Converting Enzyme; β2-AR: β2-Adrenergic Receptor; IL-4: Interleukin 4; IL-13: Interleukin 13; IL-1β: Interleukin 1β; LT-α: Lymphotoxin-α; RANTES: Regulated upon Activation, Normal T cell Expressed and Secreted; TNF-α: Tumor Necrosis Factor-α; TGF-β1: Transforming Growth Factor β1.The authors declare that they have no competing interests.HF designed the study, provided resources, coordinated the study and directed its implementation; XBL, YGZ and JZ searched the publications, extracted the data and wrote the materials and methods, results; YLX wrote the discussion and checked all data, JH was responsible for data synthesis, CT and CH helped designed the study's analytic strategy, YD edited the manuscript, YYY wrote the introduction. All authors read and approved the final manuscript.Begg's funnel plots for publication bias in selection of studies on asthma susceptibility genes in Chinese. Figure S1 Begg's funnel plots for publication bias in selection of studies on β2-AR -46G/A polymorphism. Figure S2 Begg's funnel plots for publication bias in selection of studies on β2-AR -79G/C polymorphism. Figure S3 Begg's funnel plots for publication bias in selection of studies on IL-4R -1902G/A polymorphism. Figure S4 Begg's funnel plots for publication bias in selection of studies on IL-4R -223G/A polymorphism. Figure S5 Begg's funnel plots for publication bias in selection of studies on IL-4 -589C/T polymorphism. Figure S6 Begg's funnel plots for publication bias in selection of studies on TNF-α -308A/G polymorphism. Figure S7 Begg's funnel plots for publication bias in selection of studies on FcεRIβ -6843G/A polymorphism. Figure S8 Begg's funnel plots for publication bias in selection of studies on FcεRIβ -109C/T polymorphism. Figure S9 Begg's funnel plots for publication bias in selection of studies on ACE D/I polymorphism. Figure S10 Begg's funnel plots for publication bias in selection of studies on IL-13 -2044A/G polymorphism. Figure S11 Begg's funnel plots for publication bias in selection of studies on IL-13 -1923C/T polymorphism. Figure S12 Begg's funnel plots for publication bias in selection of studies on IL-1β-511C/T polymorphism. Figure S13 Begg's funnel plots for publication bias in selection of studies on LT-α +252A/G polymorphism. Figure S14 Begg's funnel plots for publication bias in selection of studies on TGF-β1 -509C/T polymorphism. Figure S15 Begg's funnel plots for publication bias in selection of studies on CD14 -159C/T polymorphism. Figure S16 Begg's funnel plots for publication bias in selection of studies on ADAM33 T1-C/T polymorphism. Figure S17 Begg's funnel plots for publication bias in selection of studies on RANTES -28G/C polymorphism. Figure S18 Begg's funnel plots for publication bias in selection of studies on RANTES -403A/G polymorphismClick here for file


The events required to initiate host defenses against invading pathogens involve complex signaling cascades comprised of numerous adaptor molecules, kinases, and transcriptional elements, ultimately leading to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α). How these signaling cascades are regulated, and the proteins and regulatory elements participating are still poorly understood.We report here the development a completely random short-hairpin RNA (shRNA) library coupled with a novel forward genetic screening strategy to identify inhibitors of Toll-like receptor (TLR) dependent proinflammatory responses. We developed a murine macrophage reporter cell line stably transfected with a construct expressing diphtheria toxin-A (DT-A) under the control of the TNF-α-promoter. Stimulation of the reporter cell line with the TLR ligand lipopolysaccharide (LPS) resulted in DT-A induced cell death, which could be prevented by the addition of an shRNA targeting the TLR adaptor molecule MyD88. Utilizing this cell line, we screened a completely random lentiviral short hairpin RNA (shRNA) library for sequences that inhibited TLR-mediated TNF-α production. Recovery of shRNA sequences from surviving cells led to the identification of unique shRNA sequences that significantly inhibited TLR4-dependent TNF-α gene expression. Furthermore, these shRNA sequences specifically blocked TLR2 but not TLR3-dependent TNF-α production.Thus, we describe the generation of novel tools to facilitate large-scale forward genetic screens in mammalian cells and the identification of potent shRNA inhibitors of TLR2 and TLR4- dependent proinflammatory responses. Mammalian Toll-like receptors (TLRs) are single-spanning membrane proteins that display a conserved cytoplasmic Toll−interleukin 1 (IL-1) receptor (TIR) domain motif As a result of shared, cytoplasmic TIR domains, all TLRs utilize similar signaling pathways. In the MyD88-dependent pathway, recruitment of the adaptor protein MyD88 leads to the production of proinflammatory cytokines, such as TNF-α, through the sequential activation of intracellular signaling molecules such as IL-1R-associated kinase (IRAK1) and TNFR-associated factor 6 (TRAF6) Full activation of TLR signals is essential for the elimination of invading pathogens Short hairpin RNA (shRNA) mediated loss-of-function screens in mammalian cells are powerful tools for the discovery of novel gene functions. To date, two general strategies to develop shRNA libraries have been described. The first requires the synthesis of individual shRNAs targeting each gene of the genome We report the generation of a completely random shRNA library and a novel reporter cell line for the efficient identification of shRNAs with the desired phenotype. Using these tools, we performed a large-scale genomic screen to identify shRNA sequences which inhibit LPS-induced TNF-α production.- klenow fragment, which has strong strand displacement activity and lacks exonuclease activity, and a poly-A oligonucleotide, which can bind to the poly-thymidine tract of the oligonucleotide  (data not shown). This result implies that about 77% of the library contains the appropriate shRNA construct, indicating a functional library size of approximately 1.5×10To identify shRNAs which inhibit LPS induced proinflammatory signals, we generated a murine macrophage reporter cell line (RAW 264.7) that ectopically expresses the diphtheria toxin A (DT-A) fragment driven by the murine TNF-α promoter . Thus, c2 operator sites in the TNF-α promoter and used GFP fluorescence as readout for TNF-α promoter activity . Thus, we used the attenuated form of DT-A (Tox-176) which has been shown to be 10–100 fold less active than the wild type DT-A activity . Cotransactivity . Importaactivity .in vitro with kinetics similar to those of the parental RAW 264.7 cells (data not shown).Isolation of macrophage clones stably expressing both the attenuated form of DT-A (Tox-176), under the control of a Tet-repressor sensitive TNF-α promoter, and the Tet-repressor (hereafter referred to as TNF-Tox cells) proved to be the most efficient, as these clones were highly sensitive to LPS-induced cytotoxicity and grew To determine if downregulation of a known component of the TLR signaling pathway would inhibit LPS-induced cytotoxicity, we infected TNF-Tox cells with lentivirus expressing an shRNA targeting MyD88, a critical adaptor molecule required for LPS-dependent proinflammatory signals. Indeed, downregulation of MyD88 in TNF-Tox reporter cells resulted in a 3-fold increase in cell viability compared to mock infected cells . The expThe random shRNA library was transduced into the TNF-Tox reporter cell line at a concentration of virus chosen to ensure an average of one copy of shRNA per cell. Seventy two hours post-infection, the cells were stimulated with LPS and doxycycline and monitored for survival. The genomic DNA from surviving cells was harvested and PCR was used to amplify the enriched shRNA sequences. Amplified DNAs were re-cloned into the lentiviral vector and used to make fresh viral stocks containing the enriched shRNA sequences. These sequences were subsequently transduced into fresh TNF-Tox cells and the selection procedure was repeated. Following five rounds of enrichment, we observed a significant increase in cell survival compared to TNF-Tox cells infected with a scrambled non-specific shRNA sequence (data not shown). Again, PCR was used to amplify the shRNA sequences from genomic DNA. The amplified DNAs were cloned into the lentiviral vector and 140 clones were sequenced .Sequence analysis revealed that approximately half of the total shRNAs sequenced were present at least twice, indicating a significant enrichment as a result of the screening procedure . Of partTo determine if these shRNA sequences targeted genes necessary for the induction of proinflammatory cytokines, we generated lentiviruses containing these hairpin sequences and tested them individually in the TNF-Tox reporter cells. Following LPS stimulation, all four shRNA sequences significantly inhibited LPS-induced DT-A production as determined by increased reporter cell viability . InteresTo test if the recovered shRNA sequences also inhibited LPS-induced TNF-α gene expression, HP-A and HP-B were transduced into the parental macrophage-like cell line RAW 264.7. Consistent with results from the TNF-Tox reporter cell, both HP-A and HP-B significantly inhibited TNF-α gene expression in RAW 264.7 cells compared to cells infected with a non-specific shRNA sequence . The levTLR4 is unique in that its activation can promote TNF-production through both the MyD88-dependent pathway and the MyD88-independent TRIF pathway Individual TLRs initiate distinct cellular responses. Yet the mechanisms by which these specific signaling cascades are activated remain unclear. The strong association between dysregulation of TLR signaling and the development of chronic inflammatory diseases One of the hurdles in large-scale gene knockdown experiments is the development of a robust screening system to identify shRNAs with the intended effect. The need for a robust screening system becomes increasingly important as the size of the shRNA library increases, since the majority of shRNAs in a given shRNA library pool will most likely not provide the desired phenotype. Many RNA interference screens rely on the ability of an interfering RNA to rescue a cell from death caused by some cytotoxic drug. However, this approach is very limited as most signaling pathways do not end with cell death. An alternative approach is to use oligonucleotide microarrays to identify the relative enrichment or depletion of individual shRNAs in a large pool (barcode screening) In the present study, we have developed a simple positive selection approach for identifying essential components of signaling pathways, such as TLR signaling, where ligand binding does not alter cell viability. By placing DT-A production under the control of the TNF-α promoter, we were able to positively select shRNA sequences which inhibited LPS induced TNF-α production. Positive selection results in the complexity of the shRNA library pool decreasing significantly after each round of selection, thereby increasing the likelihood of identifying shRNA sequences that inhibit the targeted pathway. Importantly, this DT-A reporter system can be modified for use with other promoter systems and should greatly facilitate the identification of participants of various signaling pathways.RNAi was initially considered to be a sequence-specific gene silencing mechanism that required a perfect match between the guide strand of the siRNA and the mRNA target sequence Although verification of the siRNA target gene can be easily done by generating multiple siRNAs targeting other regions of the gene, this strategy becomes problematic if the phenotype caused by the siRNA is mediated strictly by an “off-target” effect. A recent example of this is a study by Lin et al. in which all of the top candidate siRNAs from their siRNA screen were found to elicit off-target gene silencingRecently, it has been shown that overexpression of Argonaute-2 (Ago2) can enhance the specificity and potency of shRNAs for mRNA targets with perfectly matched binding sites In summary, we have developed a completely random shRNA library and a novel reporter cell line for large-scale genomic screens. Importantly, any RNA species could potentially be targeted using this shRNA library  in virtually any mammalian cell. The use of such random shRNA libraries for gene discovery coupled with stringent selection systems can facilitate the rapid identification of small RNA modulators involved in various biological processes.5′-GGAAAGGACGAAACACC-3′) followed by a “G,” the transcriptional start point of the human U6 promoter, 18 randomized oligonucleotides, a loop sequence (5′-TTCAAGAGA-3′) TTCG loop sequence (underlined) (TTCGTTAAGTCATTGTGGAGTATCTGGAGCTATGCATCTCTTGAA-3′5′-TTCAAGAGATGCATAGCT CCACATACTCCAGAATGACTTAA) was purchased from Integrated DNA Technologies   .The procedure for the production of the shRNA library is shown schematically in 5′-GGTGTTTCGTCCTTTCC-3′) complementary to the U6 sequence of the long oligonucleotide was also added to the mixture to stop transcription by T4 DNA polymerase in the following step.300 picomoles of the 120 base pair oligonucleotide was denatured at 95°C for 10 minutes in 50 µl of NEB #2 restriction enzyme buffer  and slowly cooled at a rate of −1°C/min to allow formation of the hairpin structure of the oligonucleotide. Three nanomoles of U6 blocker primer (The gap between the 3′ end of the oligonucleotide and the 5′ end of the U6 blocker was filled using T4 DNA polymerase (New England Biolabs), which does not have strand displacement activity, at 37°C for 30 minutes in the presence of 2 mM dNTP.To remove the short U6 blocker and purify the gap-filled oligonucleotide, the reaction mixture from step B was resuspended in 1x PAGE loading buffer , 0.04% xylene cyanol FF, 40 mM Tris pH 8.0, 0.001 mM EDTA) and subjected to denaturating Urea PAGE. The DNA band of the gap-filled oligonucleotide was excised from the gel and purified.A poly-thymidine tail was added at the end of the gap-filled oligonucleotide using terminal transferase (NEB) in the presence of 1 uM dTTP according to the manufacturer's instruction. This poly-T tail functions as a termination sequence for RNA polymerase.AGCUATAGTTTAGCGGCCGCTTATACTACTCAAAAAAAAAAAAAAAAAA) according the manufacturer's instructions. Following agarose gel electrophoresis the PCR fragment was gel purified using QIAquick gel extraction kit  and resuspended in 60 µl of water for use in the subsequent PCR amplification.The resulting oligonucleotide was purified using urea polyacrylamide gel electrophoresis and used as a template to generate double stranded DNA using exonuclease deficient Klenow fragment (New England Biolabs) and the primer, dsHP  and HP-lo primer (5′-Biotin-GCC/deoxyU/ATAGTTTAGCGGCCGCTTATACTACTCAAAAAAAAAAAA AAA-3′) mixed with 2.5 units of Taq DNA polymerase . PCR was performed under the following conditions: 2 minutes at 95°C, 30 seconds at 50°C, and 30 seconds at 72°C. To avoid formation of nonspecific bands, the optimal number of PCR cycles was determined empirically.Next the DNA was amplified using 1 µl of DNA template from step 5, 3 mM dNTP, 3 mM MgClTo generate cohesive ends, the amplified PCR product was treated with the USER enzyme (New England Biolabs) according to the manufacturer's instructions, which specifically removes deoxyuridine in the DNA. The final product contains unique 7 bp and 4 bp overhangs at each end, which were used to facilitate cloning into the lentiviral vector pLL3.7 (kindly provided by Dr. Luk Van Parijs).TTCAAGAGA loop sequence, the 19-mer sequences complementary to the random sequence, followed by a poly-T termination sequence.Finally, samples were digested with BpmI to remove the extra sequences between the inverted repeat sequences, blunt-ended and religated to generate the final product which contained the following: the human U6 promoter including the “G” transcription start site, a random 18-mer sequence, a RAW 264.7 is a murine macrophage cell line, kindly provided by Dr. Genhong Cheng . RAW 264.7 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Escherichia coli LPS (serotype O55:B5) and doxycycline were obtained from Sigma , polyinosinic:polycytidylic acid (poly (I:C)) was obtained from Amersham Biosciences  and Pam3Cys was obtained from Alexis Biochemicals . The following concentrations were used for stimulation: poly (I:C) , LPS , and Pam3Cys .2 sites were inserted by PCR into each of the three reporter plasmids 10 bp downstream of the TNF-promoter TATA box to generate TNF-TetO2-DTA, TNF-TetO2-Tox176, and TNF-TetO2-EGFP.The murine TNF-α promoter fragment was isolated from the plasmid TNF-Luc 6 cells) were plated overnight in a 60 mm dish. The following day, the cells were transfected with 16.5 µg of reporter plasmid using Superfect reagent (Qiagen) according to the manufacturer's instructions. Two days later, the transfected cells were seeded in 96-well plates and selected for stable expression by 2.5 mM histidinol (Sigma). Histidinol resistant cells were cloned by limiting dilution and stimulated with LPS (1 µg/ml) to assay for DT-A induced cytotoxicity or EGFP fluorescence, as measured by CellTiter 96 AQueous (MTS) solution  or flow cytometry, respectively.RAW 264.7 cells  resistant cells were cloned by limiting dilution and assayed for DT-A induced cytotoxicity or EGFP fluorescence as above in the presence or absence of doxycycline (1 µg/ml).The generation of the tetracycline-inducible reporter plasmids required two rounds of stable transfection. First, RAW 264.7 cells were transfected with pcDNA6/TR (Invitrogen) using Superfect transfection reagent, as above, and selected for stable expression by G418 (1 mg/ml). Antibiotic resistant clones were assayed for Tet-repressor expression by transfecting individual clones with TNF-TetO5′-ATCGATTCTAGAAAGGTCGGGCAGGAAGAGGG-3′ and 5′-ATCGATCTCGAGAAAAATTCTCCGAACGTGTCACGTCTCTTGAACGTGACACGTTCGGAGAACGGTGTTTCGTCCTTTCCACAAG-3′ were used to generate a scrambled non-specific Control shRNA. Primers 5′-ATCGATTCTAGAAAGGTCGGGCAGGAAGAGGG-3′ and 5′-GATCTCGAGAAAAAGCCAGCGAGCTAATTGAGAAACTCGAGTTTCTCAATTAGCTCGCTGGCCCGGTGTTTCGTCCTTTCCACAAG-3′ were used to generate the MyD88 specific shRNA.The viral backbone of our lentiviral expression constructs is pLL3.7. PCR was performed to generate an shRNA expression cassette containing the human U6 promoter, the specific shRNA target sequence , followed by a poly-T termination sequence flanked by the restriction sites XbaI and XhoI. The PCR fragment was digested with XbaI and XhoI, and then inserted into the unmodified pLL3.7. Primers 7 and 108 transducing units/ml on RAW 264.7 cells and 293T cells, respectively, as determined by flow cytometry.Lentiviruses were produced by cotransfecting HEK-293T cells with the shRNA library lentivirus expression plasmid, the HIV-1 lentiviral packaging constructs pRSV-Rev and pMDLg/pRRE, and the VSV-G expression plasmid pMD2G, kindly provided by Dr. Didier Trono , using the calcium phosphate transfection method. Lentiviral supernatant was harvested at 48 and 72 hours post-transfection, passed through a 0.45-µm filter, ultracentrifuged for 90 minutes at 19,400 rpm in a SW28 rotor, resuspended in 200 µl of 10% CS in DMEM, and stored at −80°C until use. The infectious titer was approximately 107 cells/dish) were seeded overnight in 150 mm plates and transduced the following day with sufficient lentiviral particles from our shRNA library to infect 80% of the cells as determined by flow cytometry. Following a 72 hour incubation, transduced cells were harvested, re-seeded (1.5×107 cells/dish) in 150 mm plates, and treated overnight with doxycycline (1 µg/ml). The following day, cells were washed and stimulated with 1 µg/ml LPS in the presence of doxycycline (1 µg/ml) and incubated for 5–8 days to allow expansion of surviving clones.TNF-Tox reporter cells . PCR amplification of the shRNA inserts was performed with Platinum Taq DNA polymerase (Invitrogen) using the following primers: 5′-ATGAGCACAGAAAGCATGATC-3′; TNF-down – 5′-TACAGGCTTGTCACTCGAATT-3′; GAPDH-up – 5′ GTTGCCATCAATGACCCCTTCATTG-3′; GAPDH-down – 5′-GCTTCACCACCTTCTTGATGTCATC-3′.RNA was isolated from RAW 264.7 cells using TRIzol Reagent (Invitrogen), and cDNA was synthesized using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen). SYBR Green reactions were conducted with the IQ SYBR Green mix . Reactions were run on the MJR Opticon Continuous Fluorescence detector (Bio-Rad) and analyzed with Opticon Monitor Software 1.08 (Bio-Rad). The relative quantities of the gene tested per sample were calculated against GAPDH using the ΔΔC(T) formula. The following primers were used: TNF-up –


Before furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin) which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme's N-terminus had to be ejected from the catalytic cleft.107↓Asp108, but not at both the primary and the secondary  cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108. These correctly processed furin mutants, however, remained self-inhibited by the constrained N-terminal sequence which continuously occupied the S′ sub-sites of the catalytic cleft and interfered with the functional activity. Further, using the in vitro cleavage of the purified prodomain and the analyses of colon carcinoma LoVo cells with the reconstituted expression of the wild-type and mutant furins, we demonstrated that a three-step autocatalytic processing including the cleavage of the prodomain at the previously unidentified Arg-Leu-Gln-Arg89↓Glu90 site, is required for the efficient activation of furin.We constructed and analyzed the profurin mutants in which the egress of the emerging enzyme's N-terminus from the catalytic cleft was restricted. Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-ArgCollectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms. In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases. Specific processing is required to transform these proproteins into biologically active proteins in vitro and in cell-based tests. The original α1-antitrypsin serpin is the natural inhibitor of neutrophil elastase 355-Ile-Pro-Arg358-) in its reactive site loop and is a 0.5 nM inhibitor of furin No natural protein inhibitors of furin are known. The highly potent synthetic peptidic inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) and α1-antitrypsin variant Portland are used to inhibit furin in cleavage reactions de novo synthesized furin occurs through a two-step autoproteolytic mechanism leading to the cleavage of its cognate N-terminal prodomain. Only a few landmarks of furin activation are known. The folded furin zymogen initially undergoes an autocatalytic excision at the primary Arg-Thr-Lys-Arg107↓Asp108 site 75↓Ser76 site which releases the prodomain fragments It has been suggested that the activation of the We hypothesized that to render the emerging enzyme functionally active its N-terminal region had to egress from its original position in the preformed catalytic cleft of profurin. This event is in addition to the autocatalytic removal of the inhibitory prodomain. We also suspect that exhaustive proteolysis of the liberated prodomain is also required to prevent re-inhibition of the emerging, activated furin.in silico modeling determined that an ejection mechanism is necessary to accomplish the exodus of the N-terminus from the active site of the emerging enzyme. If this mechanism is inactivated and the N-terminus is not released from the catalytic cleft, profurin will be processed but the resulting enzyme will remain self-inhibited.To support our hypothesis, we constructed and analyzed mutant furins with amino acid substitutions and insertions into the N-terminal region of the emerging enzyme and re-examined the proteolytic processing of the furin prodomain. Collectively, our biochemical experiments and 107↓Asp108 cleavage site was positioned in the active site of profurin in a manner similar to the dec-RVKR-cmk inhibitor in the 1P8J crystal structure of murine furin In our modeled profurin structure, the relative position of the prodomain and the catalytic domain was similar to that reported for the subtilisin-propeptide complex (Protein Data Bank code 1SCJ). The identified helix sheet interaction of the propeptide with the catalytic domain is conserved in the subtilisin family 107↓Asp108 is located directly in the active site of the latent profurin. Through its electrostatic interactions with the calcium ion Asp115 plays a primary role in the active furin conformation by supporting the entire structural organization of the N-terminal Asp108-Val-Tyr-Gln-Glu-Pro-Thr-Asp-Pro-Lys117 region. There are also multiple hydrophobic interactions of Pro113 with the neighboring Val213, Phe118 and Trp138 residues. Two additional charge contacts (Glu112-Lys135 and Lys117-Glu362) also play an important role in the stabilization of the N-terminal 108–117 region. Both the Glu112-Lys135 and Lys117-Glu362 interactions appear to stabilize the structure of the N-terminal region of the active furin molecule  and will lead to the mature furin conformation where Asp108 is 40Å away from the active site. In the furin zymogen, the egress of the emerging enzyme's N-terminal region from the active site follows the autocatalytic cleavage of the primary cleavage sequence Arg-Thr-Lys-Arg107↓Asp108. To accomplish this egress, the unwinding of the 108–117 region structure is necessary. This unwinding includes a change of the backbone conformation of Lys117, the abrogation of the electrostatic contact of Asp115 with the calcium ion and also with other auxiliary electrostatic contacts and the hydrophobic interactions involving Pro113. It is highly probable that the autocatalytic cleavage at the Arg-Thr-Lys-Arg107↓Asp108 site causes Asp115 to become available for electrostatic contact with the calcium ion and, as a result, reposition the backbone to its location observed in active furin. To accommodate the ligation of Asp115 to the calcium ion, the chain reverses direction and ejects the emerging N-terminus thus liberating the multiple S′ sites of the active site of the mature, processed, furin. A similar mechanism was earlier suggested for the maturation of subtilisin BPN' According to our modeling, the zymogen conformation leading to self-cleavage provides less favorable contacts for all charged residues. The electrostatic forces originating from broken ion bridges provide the energy that would be released as a result of the Arg-Thr-Lys-Arg107↓Asp108 will access the active site without breaking the electrostatic interactions of Asp115 with the calcium ion . To confirm that the observed effects were not unique for the Gly-containing sequences, both the positively charged His6 and the negatively charged Asp-Asp-Asp-Asp-Lys sequences were also inserted between Pro113 and Thr114 . To determine the role of the hinge motion Lys117, the K117P mutant was also isolated. As a control, we constructed the catalytically inert D153N mutant with the substitution of the essential active site Asp153  that corresponded to mature furin was identified, thus confirming that the WT and the mutants were processed through an autocatalytic activation pathway involving the cleavage of profurin at the conventional primary Arg-Thr-Lys-Arg107↓Asp108 site To confirm the conversion of profurin into a correctly processed, mature furin, the WT, K117P and G6 purified samples were each subjected to N-terminal microsequencing. Only one N-terminal peptide sequence (D2)-Asp-OH -Asp-OH  and Pyr-196 site To corroborate our observations, we tested the ability of the mutants to specifically cleave anthrax protective antigen-83 (PA83), a target that is highly sensitive to furin proteolysis Normally, α1-PDX forms a covalent, SDS- and heat-stable, complex with functionally active furin We next employed ELISA to analyze, in greater detail, the ability of furins to form a complex with α1-PDX . The wel108VYQEPTDPKFPQQWYLSGVTQR130 peptide was 10–20 times more efficient in WT furin compared to that in the K117P mutant. To the contrary, the efficiency of the release of the C-terminal peptides including G498DLAIHLVSPMGTR512, the peptide from the central part of the furin sequence (M226LDGEVTDAVEAR238) and many addition tryptic peptides were highly similar in the WT and K117P furins  remained intact in the mutants.To exclude the possibility that the low proteolytic activity was due to the presence of the residual levels of the inhibitory prodomain in the furin samples, we evaluated the WT and K117P, G5 and G6 purified samples by Western blotting with the MON-150 antibody against the prodomain . The WT An additional analysis of the fractions eluted from the metal-chelating column in the course of the purification of the K117P mutant demonstrated that the prodomain was co-purified with the mature mutant enzyme . The pro75↓Ser76) in the prodomain of the G6 sample and inactivated its immunoreactivity . In contrast to the expected single cleavage of the 13 kDa tagged prodomain at the Arg-Gly-Val-Thr-Lys-Arg75↓Ser76 that should result in two peptide products To gain additional insight into the proteolytic pathway that prevents re-inhibition of furin by the residual amount of its inhibitory prodomain , we re-ein trans . Because89↓Glu90) in the autocatalytic processing of profurin, we prepared the full-length WT furin and also the constructs in which the primary (R107G), the secondary (K74G/R75G) and the tertiary (R89G) cleavage sites were each inactivated by mutations. We also constructed the double  and the triple (K74G/R75G/R89G/R107G) furin mutants in which two and three cleavage sites, respectively, were inactivated by mutations. In addition, we prepared the full-length inert D153N furin. The constructs were transiently transfected in colon carcinoma LoVo cells. We specifically selected LoVo cells for these studies because of the two frameshift mutations in the furin gene of these cells do not allow them to produce furin 89↓Glu90) was inactivated, was significantly reduced compared to that of WT furin. The inert mutant (D153N), the primary (R107G) and the secondary (K74G/R75G) cleavage site mutants as well as the double and triple mutants  were incapable of self-activation and, as a result, only the profurin form was detected in the corresponding cell lysates (89↓Glu90) and a three-step processing is required for the efficient autocatalytic processing and inactivation of the furin inhibitory prodomain in vivo. We suggest that this extensive, three-step processing helps the emerging furin to escape re-inhibition by the residual levels of the non-covalently associated prodomain.To confirm the importance of the cleavage at the tertiary cleavage site  and secondary  sites. Based on our results, we now believe that a three-step processing of the auto-inhibitory prodomain is required for its inactivation and for preventing re-inhibition of the emerging furin enzyme by the residual amounts of the non-covalently associated inhibitory prodomain. These results add another level of complexity to the molecular mechanics that are involved in the stepwise autocatalytic processing of the prodomain and imply a role for the auxiliary proteins and associated factors in these mechanisms Our Collectively, our results describe the events leading to the activation of furin and shed additional light on the restrictive role which is played by the enzyme's N-terminal region in the autocatalytic activation mechanisms of this and potentially other self-activated proteinases.Spodoptera frugiperda), Cellfectin Reagent and a pcDNA 3.1 Directional TOPO Expression Kit were obtained from Invitrogen. The baculovirus transfer vector pAcGP67-A and the BaculoGold Linearized DNA were from BD Biosciences-Pharmingen. The Fugene HD transfection reagent was from Roche Diagnostics. Sf9 insect cells were routinely grown in Sf-900 II SFM medium supplemented with penicillin-streptomycin  and Fungizone/amphotericin B . The murine MON-148, MON-150 and MON-139 monoclonal antibodies against the catalytic domain, the prodomain and the cytoplasmic tail of furin, respectively, were from Axxora. The rabbit α-tubulin antibody was from Cell Signaling Technology. The peroxidase-conjugated donkey anti-mouse and anti-rabbit IgG were from Jackson ImmunoResearch Laboratories. The TMB/M and TMB/E substrates were purchased from Chemicon.Reagents were purchased from Sigma-Aldrich unless indicated otherwise. The fluorescent substrate pyroglutamic acid-Arg-Thr-Lys-Arg-methyl-coumaryl-7-amide was obtained from Peptides International. The internally quenched fluorogenic peptide substrate Abz-Arg-Val-Lys-Arg-Gly-Leu-Ala-Tyr(NO2)-Asp-OH and the furin inhibitor dec-RVKR-cmk were from Bachem. Anthrax protective antigen-83 (PA83) was purchased from List Biological Laboratories. FLAG-tagged furin inhibitor α1-anti-trypsin variant Portland (α1-PDX) was from Affinity BioReagents. Restriction enzymes were from New England Biolabs. Sf9 insect cell line  and then C-terminally tagged with a Gly3-Ser-Gly3 linker followed by a His6 tag. The 5′-TGATAGCCCGGGCAGGGCCAGAAGGTCTTCACCAACACGTGGGCTGTGCGCATC-3′ and the ATGGTGATGATGATGGTGACCTCCCCCAGAACCTCCCCCGGCGGTGCCATAGAGTACGAG-3′5′-AGGTATACACGCGGCCGCTTA oligonucleotides were used as direct and reverse primers, respectively, in the PCR reactions .Furin was amplified by PCR from a Marathon-Ready cDNA library (Clontech) with the CCGTTTCCTCAGCAGTGGTACCTG-3′5′-TACCAGGAGCCCACAGACCCC and CGGGGGGTCTGTGGGCTCCTGGTA-3′5′-CAGGTACCACTGCTGAGGAAA forward and reverse primers, respectively. The D153N catalytically inert furin (furin D153N) with the substitution of the essential active site Asp153 was generated using the AACGATGGCATCGAGAAGAACCAC-3′5′-GGCATTGTGGTCTCCATTCTG and GTTCAGAATGGAGACCACAATGCC-3′5′-GTGGTTCTTCTCGATGCCATC forward and reverse primers, respectively.In the primers shown below the mutant positions are underlined. The K117P mutant (furin K117P) was generated by PCR mutagenesis using the GGCACAGACCCCAAGTTTCCTCA-3′5′-GGGACGTGTACCAGGAGCCC and GCCGGGCTCCTGGTACACGTCCC-3′5′-TGAGGAAACTTGGGGTCTGT oligonucleotides as the forward and reverse primers, respectively. The GGCGGTGGCGGTGGCACAGACCCCAAGTTTCCTCA-3′5′-GGGACGTGTACCAGGAGCCC (forward) and the GCCACCGCCACCGCCGGGCTCCTGGTACACGTCCC-3′5′-TGAGGAAACTTGGGGTCTGT (reverse) primers were used to obtain the G5 mutant (G5-furin). The GGTGGCGGTGGCGGTGGCACAGACCCCAAGTTTCCTCA-3′5′-GGGACGTGTACCAGGAGCCC (direct) and the GCCACCGCCACCGCCACCGGGCTCCTGGTACACGTCCC-3′5′-TGAGGAAACTTGGGGTCTGT (reverse) primers were used to prepare the G6 mutant (G6-furin). The His6 mutant (H6-furin) was constructed using the CATCACCATCATCATCACACAGACCCCAAGTTTCCTCA-3′5′-GGGACGTGTACCAGGAGCCC and GTGATGATGATGGTGATGGGGCTCCTGGTACACGTCCC-3′5′-TGAGGAAACTTGGGGTCTGT oligonucleotides as the forward and reverse primers, respectively. The GACGATGACGATAAGACAGACCCCAAGTTTCCTCA-3′5′-GGGACGTGTACCAGGAGCCC (direct) and the CTTATCGTCATCGTCGGGCTCCTGGTACACGTCCC-3′5′-TGAGGAAACTTGGGGTCTGT (reverse) primers were used to obtain the D4K mutant (D4K-furin). The resulting constructs were cloned into the XmaI and NotI sites of the pAcGP67-A vector. The authenticity of the constructs was confirmed by DNA sequencing.To construct the insertion mutants, the Gly (G), Gly-Gly-Gly-Gly-Gly (G5), Gly-Gly-Gly-Gly-Gly-Gly (G6), His-His-His-His-His-His (H6) and Asp-Asp-Asp-Asp-Lys (D4K) sequences were each inserted by PCR mutagenesis between the positions Pro113 and Thr114 of the furin sequence. The G mutant (G-furin) was constructed using the 5′-CACCATGGAGCTGAGGCCCTGGTTGCTATGGGTGGTAGCAGCAACAGGAACCTT-3′ and the 5′-TCAGAGGGCGCTCTGGTCTTT-3′ oligonucleotides as forward and reverse primers, respectively. The furin mutants with the mutations in the primary (Arg-Thr-Lys-Arg107↓Asp108), secondary  and tertiary (Arg-Leu-Gln-Arg89↓Glu90) autocatalytic cleavage sites were generated by PCR mutagenesis using the wild-type furin template. The forward GGGGACGTGTACCAGGAGCCCACA-3′5′-GTGGCAAAGCGACGGACTAAA and reverse CCCTTTAGTCCGTCGCTTTGCCAC-3′5′-TGTGGGCTCCTGGTACACGTC primers were used to construct the primary cleavage site mutant (R107G). The forward GGGGGGTCCCTGTCGCCTCACCGC-3′5′-TGGCATCGAGGAGTGACG and the reverse CCCCCCCGTCACTCCTCGATGCCA-3′5′-GCGGTGAGGCGACAGGGA primers were used to construct the secondary cleavage site mutant (K74G/R75G). The forward GGGGAGCCTCAAGTACAGTGG-3′5′-CGGCACAGCCGGCTGCAG and the reverse CCCCTGCAGCCGGCTGTGCCG-3′5′-CCACTGTACTTGAGGCTC primers were used to construct the tertiary cleavage site mutant (R89G). The double and triple mutants  in which two and three cleavage sites were inactivated by mutations were obtained by routine genetic engineering manipulations. The full-length D153N catalytically inert furin with the substitution of the essential active site Asp153 was generated using the AACGATGGCATCGAGAAGAACCAC-3′5′-GGCATTGTGGTCTCCATTCTG and GTTCAGAATGGAGACCACAATGCC-3′5′-GTGGTTCTTCTCGATGCCATC forward and reverse primers, respectively. The furin mutant constructs were cloned into the pcDNA 3.1/V5-His-TOPO and their sequences were confirmed by DNA sequencing.The full-length human furin cDNA was sub-cloned into the pcDNA 3.1/V5-His-TOPO using the 5) were transfected with 3 µl of Fugene HD reagent per 1 µg of DNA. Twenty four h after tranfection, cells were lysed in 50 mM Tris-HCl pH 8.0, supplemented with 1% SDS, 1 mM EDTA, 100 mM NaCl, 1 mM phenylmethyl sulfonylfluoride, and a protease inhibitor cocktail. The lysate samples  were analyzed by Western blotting with the furin antibodies.Human colon carcinoma LoVo cells (ATCC) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Sub-confluent LoVo cells (5×106 cell/ml) infected at a multiplicity of infection 1–2 were then cultured in suspension at 27°C in Sf-900 II SFM medium (1 L). After a 36–38 h incubation, the medium was centrifuged at 200×g and then at 15,000×g . The supernatant was concentrated 20-fold using an Amicon Ultra 30K-cutoff membrane (Millipore). 1 M Tris-HCl, pH 8.0, was added to the samples to obtain a final concentration of 20 mM. Furin was isolated from the medium samples using Ni2+-chelating chromatography on the HiTrap Chelating HP 1.6×2.5 cm size column (Amersham Biosciences) equilibrated with PBS. After eluting the impurities with 60 ml of a linear gradient of 0–25 mM imidazole, furin was eluted with 500 mM imidazole. Where indicated, 1 M NaCl was included in all of the buffers used for the purification of furin. The purified fractions were pooled, dialyzed against PBS and stored at -80°C. Purified furin samples were analyzed by SDS-PAGE, Western blotting and enzyme activity assays. The typical yield of purified furin was approximately 1 mg/liter of cell culture medium. Following SDS-PAGE and the transfer of the proteins onto a membrane support, the N-terminal peptide sequence of furin was determined by N-terminal microsequencing at ProSeq .Sf9 cells were co-transfected with the recombinant pAcGP67-A vectors and the BaculoGold linearized DNA using Cellfectin. The amplification of the recombinant baculovirus and the preparation of a high-titer viral stock were performed in Sf9 cells according to the manufacturer's instructions (Invitrogen). Cells  was co-incubated for 15 min at ambient temperature with furin  in either PBS or the Sf-900 II SFM medium. For the Western blot analysis, the concentrations of α1-PDX and furin in the reactions were 10-fold less . Furin activity was blocked by adding 5 mM EDTA and the 2× SDS-PAGE sample buffer  to the reactions. The reactions were heated for 3 min at 95°C and then analyzed by SDS-PAGE in a 4–20% acrylamide gradient gel followed by either Coomassie staining or Western blotting of the reaction aliquots with the MON-148 antibody.2SO4. The A450 value was measured using a plate reader. The samples were analyzed in triplicate.To measure the efficiency of the α1-PDX binding with furin, the wells of a 96-well plate (Nunc) were coated for 16 h at 4°C with purified furin constructs (1 µg/ml in 100 µl PBS). BSA (1 µg/ml 100 µl PBS) was used as a control. The wells were then blocked with 3% BSA in PBS-0.05% Tween 20 for 2 h at the ambient temperature. α1-PDX (50 µg/ml in 100 µl PBS containing 3% BSA and 0.05% Tween 20) was added to the wells and the plates were incubated for 1 h at the ambient temperature. After washing the wells five times with PBS-0.05% Tween 20, the murine FLAG M2 antibody (0.5 µg/ml in 100 µl PBS containing 3% BSA and 0.05% Tween 20) was added to the wells and the plates were incubated 1 h at the ambient temperature. The unbound antibody was removed by washings with PBS-0.05% Tween 20. The peroxidase-conjugated donkey anti-mouse IgG  was added to the wells for 1 h. Following extensive washing with PBS-0.05% Tween 20 and then with water, a TMB/E substrate was added to the wells. The reaction was stopped by adding 0.1 ml 1 M H5′-CAGAAGGTCTTCACCAACACG-3′ and 5′-TCACCGTTTAGTCCGTCGCTTTGC-3′ oligonucleotides were used as direct and reverse primers, respectively, in the PCR reactions. The amplified fragment contained the prodomain sequence from Gln27 to the Arg-Thr-Lys-Arg107↓ primary cleavage site. The PCR fragment was subcloned into the pTrcHis A vector (Invitrogen). The resulting protein was expressed in frame with a His6 tag sequence, a linker region and an additional Xpress short tag. One Shot TOP 10 E.coli cells (Invitrogen) were transformed with the recombinant prodomain construct. The expression of the recombinant protein was induced with 1 mM isopropyl-1-thio-β-D-galactopyranoside. The collected cells were lysed, and the recombinant prodomain was purified from the cell lysates using Ni2+-chelating chromatography and reverse-phase HPLC. The purity of the recombinant prodomain construct was confirmed by SDS-PAGE and mass spectrometry.The prodomain was prepared using PCR amplification of the full-length furin cDNA. The 2 and 1 mM β-mercaptoethanol. Where indicated, dec-RVKR-cmk (50 µM) was added to the reactions. The molecular mass of the intact prodomain and the digest products was determined by MALDI-TOF mass spectrometry on a Brucker Daltonics Autoflex II TOF/TOF mass spectrometer. To determine the N-terminal peptide sequence of the cleavage products and, consequently, the scissile bond, the reactions were separated by Tricine/SDS-PAGE in a 10–20% polyacrylamide gradient gel followed by the transfer of the digest fragments onto a membrane support. The Coomassie stained bands were excised and their N-terminal sequence was determined by N-terminal microsequencing at ProSeq.The furin prodomain  was co-incubated with the purified furin samples (an enzyme-substrate ratio of 1:1 and 1:2) for 1 h at 37°C in 100 mM Hepes, pH 7.5, containing 1 mM CaClhttp://peptideprophet.sourceforge.net). Only the peptides with a probability score that exceeded 0.95 and a cross-correlation (Xcorr) value that exceeded 2.0 were further considered. The yield of the tryptic peptides was measured using the differential peptide spectral count analysis The purified WT and K117P furins (0.4 mg/ml each) were co-incubated for 5 min at 37°C in with trypsin (Promega) at a trypsin-furin ratio of 1:100 and 1:1000 (w/w) in PBS. Trypsin was then inactivated by 1 mM phenylmethyl sulfonylfluoride. The digest peptides were analyzed by LC-MS/MS using an Eksigent Nano 2D-LC system coupled with LTQ mass spectrometer (Thermo Scientific). The spectra were searched using Sorcerer SEQUEST (Sage-N Research) and filtered by PeptideProphet -Asp-OH (5 µM) was used as a substrate. The concentration of the wild-type (WT) and mutant furin in the reactions was 1 nM and 1 µM, respectively. The steady-state rate of substrate hydrolysis was monitored continuously (λex = 320 nm and λem = 420 nm) at 37°C using a fluorescence spectrophotometer. When the Pyr-Arg-Thr-Lys-Arg-methyl-coumaryl-7-amide substrate was used, the steady-state rate was monitored at λex = 360 nm and λem = 465 nm. The samples were measured in triplicate.Furin activity was measured in wells of a 96 well plate in 0.2 ml 100 mM Hepes, pH 7.5, containing 1 mM CaCl2 and 1 mM β-mercaptoethanol. The reactions were stopped by adding 2× SDS-PAGE sample buffer  to the samples. The digest reactions were analyzed in an 8–16% polyacrylamide gradient gel.PA83  was co-incubated with furin at the indicated enzyme-substrate ratio for 1 h at 37°C in 100 mM Hepes, pH 7.5, containing 1 mM CaClhttp://ffas.ljcrf.edu). The human furin propeptide was docked into the crystal structure of the mouse furin catalytic domain using the DOT program (www.sdcs.edu/CCMS/DOT). To avoid steric clashes the several side chain positions were corrected manually using the Xfit program . The structure was then energy-minimized using the CHARMm22 force field molecular dynamics software of the Discovery Studio software package . The model was then validated using Molprobity (molprobity.biochem.duke.edu).The structure of the human WT and mutant profurin was modeled using the available coordinates of the highly homologous (97% identity) catalytic domain and P-domain of murine 108–574 furin (Protein Data Bank code 1P8J) and the prodomain of murine 26–107 PC1  as templates, and the Modeler software (Table S1(0.05 MB DOC)Click here for additional data file.


MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects.Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184.We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer. MYCN transcription factor is the single most important prognostic indicator of poor patient survival and determination of genomic MYCN copy number status plays a major role in the stratification of patients for treatment [Neuroblastoma is a paediatric cancer of the sympathetic nervous system and accounts for approximately 15% of all childhood cancer related deaths. The disease has a highly varied clinical outcome, some tumours can spontaneously regress without treatment, while others can progress and lead to the death of the patient in spite of intensive multi-modal chemotherapy. Amplification of the reatment , and morreatment -6.MicroRNAs are a class of small 19-25 nt) noncoding regulatory RNAs that regulate gene expression through their binding to sites within the 3'UTR of an mRNA target gene, causing either mRNA degradation or translational inhibition . These s-25 nt noAKT2 mRNA, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, an important pro-survival pathway in cancer [Through miRNA expression profiling of different genetic subtypes of neuroblastoma, Chen and Stallings  and othen cancer -23. ThusNeuroblastoma tumour samples were obtained from patients at Our Lady's Hospital for Sick Children in Crumlin, Ireland or through the Children's Oncology Group (USA) and have been previously described in aCGH , mRNA 2 and miRNKelly and SK-N-AS cell lines were purchased from the European Collection of Animal Cell Cultures . SHEP-TET21 cells were obtained from Dr. Louis Chesler with permission of Prof. Manfred Schwab . Kelly cAKT2 were obtained from Applied Biosystems . Three different siRNAs against AKT2 were chosen  (s228853 sense strand ACAACUUCUCCGUAGCAGAtt and anti sense strand UCUGCUACGGAGAAGUUGUtt).Pre-miR™ and Anti-miR™ to miR-184 and negative control 1 (a scrambled oligonucleotide) were obtained from Ambion . Short interfering (si)RNAs targeting AKT2 were introduced into the cells by reverse transfection using the transfection agent siPORT™ NeoFX™(Ambion). Cell culture media was changed after 8 hours to remove the transfection reagent in an attempt to avoid toxicity which may be caused by NeoFX™. Total RNA/miRNA was extracted 24, 48 and 72 hours after transfection using RNeasy Kit/mirNeasy© kit .The Pre-miR™ and Anti-miR™ to miR-184, negative control 1 and the siRNAs to -ΔΔCT).Reverse transcription was carried out using 50 ng of total RNA with the primer specific for miR-184 and the TaqMan microRNA reverse transcription kit (Applied biosystems). qPCR was carried out on the 7900 HT Fast Realtime System (Applied Biosystems). RNU66, a small RNA encoded in the intron of RPL5 , was used for normalization in miRNA studies and RPLPO ribosomal protein was used for normalization in gene expression studies . A relative fold change in expression of the target miRNA/gene transcript was determined using the comparative cycle threshold method . The following oligonucleotides were designed which encode the sense and antisense strands of the pre-miR184 sequence. These oligonucleotides include the appropriate 5' and 3' overhangs to facilitate cloning into the linearised pcDNA6.2-GW/EmGFP vector .Pre-miR184 Sense strand:TGCTGCCAGTCACGTCCCCTTATCACTTTTCCAGCCAGCTTTGTGACTGTAAGTGTTGGCAGGAGAACTGATAAGGGTAGGTGATTGAPre-miR184 Antisense strand:CCTGTCAATCACCTACCCTTATCAGTTCTCCTGCCAACACTTACAGTCACAAAGCTGGCTGGAAAAGTGATAAGGGGACGTGACTGGCThe pcDNA6.2-GW/EmGFP-miRNA-184 construct or the control construct  was transfected into Kelly and SK-N-AS cells using lipofectamine 2000  according to manufacturers instructions. Quantitative real-time PCR and fluorescent microscopy were carried out to determine efficient transfection and transcription of the vector.AKT2 was obtained from Prof. Joe Testa  [The expression vector pcDNA 3 containing delphia) . 1 μg of6 cells/ml. An aliquot of 100 μl of suspension (1 × 105 cells) was stained with 5 μl Annexin-V-FITC and 5 μl PI, and incubated for 15 minutes at room temperature in the dark. Binding buffer (400 ul) was added and cells acquired  immediately using a BD LSR II flow cytometer  and analysed using BD FACSDiva 4.0 Software. Experiments were performed in multiples to qualify apoptosis by phosphatidylserine (PS) externalization.Apoptosis was demonstrated by annexin-V staining and propidium iodide (PI) exclusion using the FITC Annexin-V Apoptosis Detection Kit I . Briefly, adherent and supernatant Kelly cells were collected, washed twice in cold PBS, and resuspended in 1× Annexin-V binding buffer at a concentration of 1 × 10Cell Death was also evaluated using the 3/7 Caspase detection kit from Promega . Neuroblastoma experimental cells were plated in quadruplicate in 96-well plates. 72 hours after transfection, 10 ul of caspase 3/7 was added to each well. Samples were read after 1 hr of incubation with the caspase substrate on a Viktor Microplate luminometer .4 cells per well. When carrying out transfections using the microRNA mimics or anti-miRs (as described above) each time point was set up with a non-transfected (with transfection reagent) and a scrambled oligonucleotide control (negative 1). Cells were trypsinised from 6 well plates at 24, 48 and 72 hour time points, and re-suspended in 1 ml of media. A haemocytometer was used to count cell numbers. Counts from triplicate wells were averaged.For cell number assays, cells were set up in triplicate in 6 well plates. Cells were seeded at equal densities of 3 × 10Total protein was isolated from cells using a radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma). Protein concentration was measured using the BCA assay from Millipore. Proteins were fractionated on 10% polyacrylamide gels, and blotted onto nitrocellulose membrane. The membrane was probed with the Anti-AKT2 Antibody (Millipore) or anti-MYCN (Abcam), anti β-Actin from (Abcam) or anti GAPDH (abcam) (used for loading controls). Signal was detected using Immoblion Western (Millipore).TGACAGTTCTCCGTCCCTACTGGCCTTTCTCCTCGTCTTCGCTCAGCA 3'A 76nt long region of the 3'UTR of AKT2 containing the predicted miR-184 binding site (underlined) was ligated into the pMiR-Reporter vector (Ambion) 3' of the luciferase gene:5'CTAGTCCTCTGTGTGCGATGTTGTTATCAs a negative control, three mutations (lower case) were introduced into the seed region of miR-184 binding site of this sequence:TGACAGTTCTtCaaCCCTACTGGCCTTTCTCCTCGTCTTCGCTCAGCA 3'5'CTAGTCCTCTGTGTGCGATGTTGTTATC4 in 12 well format. After 24 hrs the pMir-Reporter containing the AKT2 binding site for miR-184 or the mutated AKT2 binding site were co-transfected with either the pre-miR-184 mimic or a scrambled negative control sequence using Lipofectamie 2000. All experiments were also co-transfected with the pmiR-Report β-galactosidase vector as a control for transfection efficiency and normalization. Luciferase activity was measured by One-Glo luciferase assay (Promega) according to manufactures instructions after 24 hours on the Viktor Plate Reader.KELLY cells were plated at 8 × 10MYCN construct which is repressible by doxycycline. As demonstrated by qPCR and Western blotting, addition of doxycycline to the cell culture caused a dramatic reduction in both MYCN mRNA and protein levels  of miR-184 predicted targets was the 3'UTR of AKT2, which had a high level of sequence identity with the miR-184 seed region, a 13 base pair match . As one might expect, AKT2 was expressed at higher levels in MNA tumors relative to non-MNA tumors (P < 0.035), as MYCN suppresses miR-184 transcript quantities  following transfection of miR-184 mimics into Kelly (P = 0.004). These results were also observed at protein level Figure  and SK-N4)Figure , and conl Figure . MiR-184AKT2 mRNA, since co-transfection of a pMir-Reporter containing the AKT2 binding site for miR-184 and mature miR-184 mimics significantly (p < 0.003) diminished luciferase activity while co-transfection of the reporter with a negative control oligonucleotide had no effect .  differences in AKT2 mRNA levels in each of the transfected cell populations at each time point, consistent with expectations. From all of the above experiments, we conclude that the phenotypic effects of miR-184, at least to a large extent, can be attributed to the targeting and reduction of AKT2.To demonstrate that the increase in cell numbers that occurred following miR-184 knockdown resulted specifically from . Figure . Since tn Figure . As illuAKT2 as an important pro-survival gene in neuroblastoma and our results further demonstrate that MYCN indirectly regulates AKT2 through miR-184. It is unknown whether MYCN directly or indirectly suppresses miR-184 expression. There are two DNA sequence motifs, GGCATG and CCCGTG, reported to bind to MYCN at the MCM4 and MCM5 loci [AKT2 mRNA from degradation by miR-184, permitting this important pathway to remain functional.This study identifies CM5 loci , approxiCM5 loci , indicatAKT2 mRNA by itself can fully account for the observed apoptotic phenotype. First, siRNA mediated inhibition of AKT2 in Kelly and SK-N-AS cells induces a level of apoptosis that is comparable to miR-184 ectopic up-regulation. Second, ectopic up-regulation of AKT2 causes an increase in cell numbers similar to that observed following miR-184 knock-down, and the effects of ectopic miR-184 up-regulation are abrogated by ectopic over-expression of an AKT2 expression plasmid lacking the miR-184 binding site. We can not rule out the possibility that the targeting of other genes by miR-184 has altered the phenotypes of these cells in some undetectable manner, only that miR-184 targeting of AKT2 fully accounts for the pro-apoptotic effects.Although miR-184 is predicted to target several hundred genes, several lines of evidence indicate that the targeting of AKT2 is a homolog of the v-akt oncogene, a protein serine/threonine kinase pro-survival protein, which is member of the AKT family of proteins  that are activated by the phosphatidylinositol 3' kinase pathway [AKT2 isoform expression levels are critical for neuroblastoma cell survival even in the absence of chemotherapeutic compounds. The other isoform which is expressed at high levels in neuroblastoma cell lines, AKT1, does not possess a miR-184 target site, remains constant in all of our experiments, and does not rescue the cells from the effects of miR-184 over-expression. This is consistent with findings that AKT2 does not share complementary functions with AKT1 regarding cell invasiveness and survival in other forms of cancer [ pathway . The pho pathway . Activat pathway . In addi pathway . Our resf cancer ,33.AKT2 kinase is frequently observed in ovarian cancer [The deregulation of the AKT signalling pathway has been associated with numerous other cancers including glioblastoma, breast, prostate and lung . The actn cancer , breast n cancer , and appn cancer . In addin cancer . AKT2 wan cancer . Moro etn cancer .SHIP2, a protein that causes a reduction in activated phosphorylated AKT, but not in total AKT amounts. Thus, miR-205, which is elevated in aggressive SCC, is acting oncogenically by targeting SHIP2, allowing AKT activation. They further report that miR-184 antagonizes miR-205, so in this sense, miR-184 is acting as a tumor suppressor. The effects of ectopic miR-184 over-expression on AKT mRNA or protein levels was not examined by Yu et al (2008), and this was the first report of a microRNA interfering with the action of another miRNA. Our results indicating that miR-184 acts in a tumor suppressive manner in neuroblastoma does not shed further light upon these seemingly contradictory reports, as the role of any miRNA in cancer is likely to be cell context dependent.There is presently not very much known about miR-184 involvement in cancer. It was reported to be up-regulated in squamous cell carcinoma (SCC) of the tongue, and suppression of this miRNA in SCC cell lines showed reduced cell numbers and an increase in apoptosis, suggesting an anti-apoptotic role for mir-184 . HoweverAKT2 mRNA, is a naturally occurring inhibitor of this protein, and has potential value in miRNA mediated therapeutics for any form of cancer dependent on AKT2.Finally, a number of studies have sought to identify small molecule inhibitors of AKT family members for cancer therapy ,43. MiR-The authors declare that they have no competing interests.NHF, IB, AT, DMM, PGB, JR carried out the experimental work, KB provided data analysis, AOM and MOS provided tumor samples, clinical information, and/or histopathological analysis, NHF, IB, JR, PGB and RLS designed the study, interpreted the findings and participated in writing the paper. All authors read and approved the manuscript.MYCN amplified), SH-EP TET21 cells untreated and treated with doxycycline. GAPDH was used as the endogenous loading control. (B) Relative MYCN mRNA levels in SHEP TET21 cells treated (MYCN off) and untreated (MYCN on) with doxycycline as assessed by TaqMan qPCR. (C) RT-qPCR analysis of miR-184 levels in SH-EP-TET21 cells treated (MYCN off) and untreated with doxycycline (MYCN on). miR-184 expression is relative to untreated SH-EP cells.(A) Western blot showing MYCN protein in Kelly  and SK-N-AS (B) cells at 24 and 48 hours following transfection with pcDNA6.2-184 and empty vector (negative control). All values are relative to the negative control at 24 hrs, set as 1.0. Growth curves for Kelly cells (C) and SK-N-AS cells (D) after transfection with the stem loop precursor sequence of miR184 cloned into pcDNA6.2-GW/EmGFP. PcDNA6.2-GW/EmGFP-miRnegative control was used as a negative control (NC). Caspase 3/7 Assay for Kelly cells (E) and SK-N-AS cells (F) after transfection with the same stem loop precursor sequence of miR184 or the pcDNA6.2-GW/EmGFP-miRnegative control, set as 1.0. FACs analysis of annexin V staining in Kelly cells transfected with pcDNA6.2-GW/EmGFP-miRnegative control (G) and pcDNA6.2-184 (H).Click here for fileAKT1 mRNA levels following transfection of Kelly cells with miR-184 mimics or negative control oligonucleotide at different time points. (B) Relative AKT1 mRNA levels following transfection of Kelly cells with three different AKT2 siRNAs or negative control siRNA at different time points. RPLPO was used as endogenous control for RT-qPCR and all values are relative to the negative control at 24 hrs.(A) Relative Click here for fileAKT2 mRNA  and protein  in Kelly cells and SK-N-AS cells, respectively, by qRT-PCR or Western blot. RPLPO was used as endogenous control for RT-qPCR and all values are relative to the negative control at 24 hrs.Assessment of siRNA knockdown of Click here for fileAKT2 mRNA levels at 24, 48 and 72 hours following transfection with different combinations of plasmids and oligonucleotides. In the AKT2 rescue experiment (AKT2 plasmid + miR-184 mimics), AKT2 levels are not significantly different from the negative controls  and are intermediate between cells transfected with AKT2 alone (cells having endogenous and ectopic AKT2) and cells transfected with miR-184 mimics alone (inhibited endogenous and no ectopic). All AKT2 mRNA levels are relative to the co-transfection with pcDNA3.1 empty vector and negative control at the 24 hr time point.qRT-PCR assessment of Click here for file


Treponema denticola harbours a genetic locus with significant homology to most of the previously characterized Treponema pallidum tro operon. Within this locus are five genes (troABCDR) encoding for the components of an ATP-binding cassette cation-transport system (troABCD) and a DtxR-like transcriptional regulator (troR). In addition, a σ70-like promoter and an 18 bp region of dyad symmetry were identified upstream of the troA start codon. This putative operator sequence demonstrated similarity to the T. pallidum TroR (TroRTp) binding sequence; however, the position of this motif with respect to the predicted tro promoters differed. Interestingly, unlike the T. pallidum orthologue, T. denticola TroR (TroRTd) possesses a C-terminal Src homology 3-like domain commonly associated with DtxR family members. In the present study, we show that TroRTd is a manganese- and iron-dependent transcriptional repressor using Escherichia coli reporter constructs and in T. denticola. In addition, we demonstrate that although TroRTd possessing various C-terminal deletions maintain metal-sensing capacities, these truncated proteins exhibit reduced repressor activities in comparison with full-length TroRTd. Based upon these findings, we propose that TroRTd represents a novel member of the DtxR family of transcriptional regulators and is likely to play an important role in regulating both manganese and iron homeostases in this spirochaete. Treponema denticola, a member of the ‘red complex’, is a motile, asaccharolytic, anaerobic spirochaete that is common to the normal oral flora of humans -type transporters specifically devoted to the transport of Fe2+, implicating the importance of this metal to the organism  is a cation-dependent transcriptional repressor possessing structural and biochemical properties distinct from its T. pallidum orthologue.Consistent with the predicted metabolic diversity of T. denticola ATCC 35405 genome sequence revealed the presence of a genetic locus demonstrating significant similarity to the T. pallidum tro operon. In T. denticola, the tro operon consists of five open reading frames: troA, troB, troC, troD and troR. This operon is predicted to encode the components of an ABC cation-transport system composed of TroA, a solute-binding protein; TroB, an ATPase; TroC and TroD, cytoplasmic membrane permeases; and TroR, a metal-dependent transcriptional regulator , TroRTd harbours a 70-amino-acid C-terminal extension.Analysis of the onserved . ResultsT. denticola troA start codon revealed the presence of a σ70-like promoter sequence and an 18 bp region of dyad symmetry  sequences differ relative to the T. pallidum tro-P/O sequences  and MntR (Bs MntR) is shown in Td is predicted to have secondary structure similar to DtxR and to contain a comparable N-terminal DNA-binding domain followed by a helical dimerization domain. Additionally, similar to DtxR, TroRTd appears to possess an extended C-terminal Src homology 3-like (SH3-like) domain that is absent from TroRTp and MntR.TroRcus MtsR . An aligTp, TroRTd demonstrates a high degree of conservation at both sites 1 and 2 with 5/7, 4/7 and 7/7 identical residues respectively (Td and TroRTp is the presence of an asparagine (N10) corresponding to DtxR M10 and MntR D8. Additionally, the C102 residue of DtxR is substituted by E102 in MntR, TroRTd and TroRTp. All four proteins are predicted to have tether regions linking the N- and C-terminal regions of the proteins; this region is designated α7 in MntR and is proline-containing in DtxR and TroRTd  is thought to stabilize dimer formation and allow metal ion binding at the primary site , which is the putative metalloregulatory site that is required for metal ion activation . In compd TroRTd . The biooRTd (Td .T. denticola troR was cloned into the pBAD/HisA expression vector resulting in pPJB114. The His-tagged protein (TroRTd-6xHis) was overexpressed in Escherichia coli, producing a 26 kDa protein that localized to the insoluble fraction of the cell lysate. Following removal of soluble proteins, TroRTd was extracted from the insoluble pellet with 0.2% Sarkosyl and purified with a Nickel-agarose affinity column as described in the Experimental procedures section. This procedure yielded TroRTd-6xHis that was purified to apparent homogeneity as determined by SDS-PAGE (data not shown) and was used for antibody production. Following numerous attempts using a variety of conditions, including those previously used to purify TroRTp  or pBAD/HisA (vector control). Immunoblot analysis of E. coli lysates showed that TroRTd was expressed from pPJB113, but not from the empty vector control (data not shown). Results demonstrated that β-galactosidase activity from strains DEN100 (harbouring pTDE100 and pBAD/HisA) and PAL100 (harbouring pPAL100 and pBAD/HisA) was high in the absence of TroRTd  supplemented with 5 μM Fe2+, Mn2+ or Zn2+. At 24 and 48 h post inoculation, RNA was harvested and troA transcript levels were quantified by qRT-PCR. Relative amounts of the troA transcripts from spirochaetes grown in the presence of cations were then compared with transcript levels from unsupplemented controls. All reactions were normalized to flaA as an internal control . β-Galactosidase assays were conducted in E. coli strains harbouring the tro-P/O lacZ-transcriptional fusion constructs, pTDE100 or pTPA100, along with plasmids expressing full-length TroRTd (strain DEN113) or various truncated derivatives as follows: TroRTdΔ157−222 (strain DEN113A), TroRTdΔ147−222 (strain DEN113B), TroRTdΔ137−222 (strain DEN113C) and TroRTdΔ127−222 (strain DEN113D). Results for the T. denticola tro-P/O region demonstrated significantly decreased β-galactosidase activity in strain DEN113 (96% repression), compared with the control strain DEN100, as expected. In contrast, expression of the truncated TroRTd proteins in strains DEN113A, DEN113B and DEN113C demonstrated increased β-galactosidase activity, corresponding to 64%, 58% and 43% repression respectively, indicating decreased repressor function. Expression of TroRTdΔ127−222, a deletion extending into the proline-containing region of the protein, in strain DEN113D resulted in maximal β-galactosidase activity (similar to DEN100), indicating virtually complete loss of repressor function , indicating intact repressor function. In contrast, TroRTdΔ127−222 expression in strain PAL113D resulted in only slightly decreased β-galactosidase activity corresponding to partial repressor activity (36%)  . Taken tTp and B. subtilis MntR are manganese-responsive and lack C-terminal SH3-like domains. In order to assess the importance of the TroRTd C-terminal domain with respect to metal specificity, β-galactosidase activity from strains DEN113A and PAL113A was measured in response to metal ion supplementation. In the presence of TroRTdΔ157−222 expression, β-galactosidase activity from both DEN113A and PAL113A decreased in response to Mn2+ and Fe2+ and also decreased slightly in response to Co2+  relaxes the specificity for Mn2+  in T. pallidum  or MntR (Mn2+) and may be more similar to DtxR, SirR and MtsR.In the present study, we identified the B. subtilis, DtxR (M10D/C102E) as predicted responded to Mn2+. However, when intracellular concentrations of Fe2+ were increased by deregulating iron uptake in a B. subtilis fur mutant, DtxR (M10D/C102E) also responded to Fe2+ (2+ (0.79 and 0.24 nmol mg−1 of protein respectively) and Fe2+ (4.2 and 3.5 nmol mg−1 of protein respectively) in E. coli and T. denticola. While there are some differences in the intracellular levels of these metals (particularly Mn) under the conditions tested, it is interesting to note that the data presented in this report, as measured in E. coli (lacZ reporter assays) and T. denticola (qRT-PCR), indicate that TroRTd responded similarly to Mn2+ and Fe2+ in both cytosolic backgrounds.Previous studies by  to Fe2+ . Thus, tTd with DxtR, MntR and TroRTp revealed the presence of conserved N-terminal helical domains as well as conserved metal binding site residues. Additionally, TroRTd had a C-terminal SH3-like domain. The importance of the C-terminal extension present on TroRTd was investigated using a number of C-terminally truncated TroR proteins. Our results demonstrated that loss of the C-terminal region of TroRTd affected repressor function at the T. denticola tro-P/O, but not at the T. pallidum tro-P/O. These results are consistent with the fact that TroRTp naturally lacks this C-terminal extension. It is possible that the differences observed in the tro-P/O regions of these two treponemes may reflect their different-sized TroRs; i.e. each species has optimized its P/O region for TroR binding. Conversely, the different-length TroRs may be produced to compensate for the altered tro-P/O regions. In any case, it appears that the TroRTd requires an extended C-terminal region for maximal repression of the T. denticola tro-P/O. Further results indicated that the proline-containing region may be necessary for TroR to function at both T. denticola and T. pallidum tro-P/O regions as a deletion affecting this region-abrogated repression. This domain may function in protein–protein interactions that may be important for subsequent DNA binding; however, this remains to be experimentally determined. The metal specificity of C-terminally truncated TroR remained unchanged, suggesting that this domain was not involved in metal binding. Similar to DtxR, the specific function of TroRTd C-terminal domain remains unclear.Secondary structure predictions and amino acid comparisons of TroRTd is a Mn2+- and Fe2+-dependent transcriptional regulator that negatively regulates expression of the T. denticola tro operon. Structural predictions show that TroRTd possesses conserved metal binding site residues as well as an SH3-like domain. Overall, these findings suggest that TroRTd is a novel member of the DtxR family of transcriptional regulators and would be predicted to play a critical role in regulating Mn2+ and Fe2+ levels in T. denticola. Further studies will be required to determine the role of the T. denticola tro operon in metal ion homeostasis in natural infections. Studies are ongoing to obtain soluble TroR for further biochemical and crystallography analyses to gain further insight into the structure and function of TroRTd.In summary, we have demonstrated that TroRE. coli strains were grown at 37°C on Difco LB-Lennox (LBL) agar or in LBL broth unless otherwise stated. For metal specificity assays, E. coli strains were grown in Difco M9 Minimal Salts supplemented with 0.5% w/v Bacto Casamino Acids and 0.2% v/v glycerol (M9CG). When appropriate, antibiotics were used at the following concentrations: 100 μg ml−1 ampicillin, 25 μg ml−1 chloramphenicol and 25 μg ml−1 kanamycin (Km).The bacterial strains and plasmids used in this study are described in Treponema denticola ATCC 35405 was grown at 37°C under anaerobic conditions  in NOS media (ATCC medium 1494) supplemented with 1% EX-CYTE  instead of rabbit serum. Growth rates in NOS-E media were found to be similar to those in NOS media. Metal-deplete NOS-E media (NOS-EC media) was prepared by chelating NOS-E media two times with 2.5% (w/v) Chelex 100 under anaerobic conditions. NOS-EC media was then supplemented with 1 mM MgSO4 and 0.1 mM CaCl2 and filter-sterilized.E. coli TOP 10 cells were transformed as per manufacturer's instructions (Invitrogen). DNA sequencing was performed by ACGT.DNA manipulations were performed using standard methods. PCR was performed using the Expand High Fidelity PCR System (Roche Applied Science). PCR and restriction enzyme-digested products were purified using a QIAquick Gel Extraction Kit (Qiagen). Plasmids were purified using a QIAprep Spin Miniprep Kit (Qiagen). Ligation reactions were performed using a Fast-Link Quick Ligase Kit (Epicentre Technologies). Chemically competent lacZ locus from pBAD/His/lacZ were PCR-amplified using the OriCm-F1/OriCm-R1 and LacZ-F2/LacZ-R2 primer pairs respectively  to facilitate annealing  encoding the troR alleles were amplified from frozen stocks of T. denticola ATCC 35405 using the following primer pairs: TdtroR-F2/TdtroR-R3, TdtroR-F2/TdtroR-RA, TdtroR-F2/TdtroR-RB, TdtroR-F2/TdtroR-RC, TdtroR-F2/TdtroR-RD and TdtroR-F2/TdtroR-RH2  and then re-suspended in NOS-EC media. NOS-EC media supplemented with 5 μM Fe2+, Mn2+ or Zn2+ was inoculated with ∼107 spirochaetes ml−1 and RNA was harvested at 24 and 48 h. qRT-PCR experiments were performed as previously described for Borrelia burgdorferi (T. denticola RNA was extracted using TRI-Reagent (Sigma) as described by the manufacturer. RNA was treated with RNase-free DNase (Qiagen) and purified using the RNeasy miniprep kit (Qiagen). SuperScript III (Invitrogen) was used to synthesize first-strand cDNA following manufacturer's instructions. qRT-PCR primers and probes specific for troA and flaA were designed using Primer Express 1.0 and are shown in m forward and reverse primers and 250 nm probe. All reactions were carried out on the ABI PRISM 7900HT Sequence Detection System (Applied BioSystems) using a PCR cycle of 2 min at 50°C, 10 min at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Each transcript was normalized by comparison with the constitutively expressed, internal control flaA  and incubated for 10 min at RT. The insoluble material was pelleted by centrifugation , re-suspended in Lysis Buffer B (B-PER) and incubated for 10 min at RT. The insoluble material was pelleted, re-suspended in Solubilization Buffer  and gently agitated for 60 min at 4°C. The remaining insoluble material was removed by centrifugation, the supernatant was filter-sterilized and allowed to batch-bind to HIS-Select Nickel Affinity Gel (Sigma) prior to loading onto a gravity fed column. The flow through was collected and applied to the column a second time, followed by washing of the column with Wash Buffer . Protein was eluted with Elution Buffer . Fractions were analysed by SDS-PAGE and those containing TroRTd were pooled, concentrated and stored in Storage Buffer . Protein concentrations were determined using a BCA protein assay kit (Pierce). Purified protein was used to raise anti-TroRTd polyclonal antiserum in female New Zealand White rabbits at Cocalico Biologicals using a standard protocol.For purification of recombinant protein, TroRi LYS114 . BacteriE. coli cultures and re-suspending the cells in 200 μl of 1× SDS-PAGE sample buffer prior to heating at 100°C for 10 min. Once cooled, 1 μl of DNase  was added to each sample to decrease viscosity. Treated lysates were then electrophoresed on 4–20% Express Gels (ISC BioExpress). Proteins were electrophoretically transferred to nitrocellulose membranes . Immunoblot analysis was performed as follows at RT: membranes were blocked with TBS-TS  for 60 min, followed by incubation for 1 h with a 1/2000 dilution of the primary antibody  in TBS-T . Membranes were washed three times with TBS-T, followed by incubation for 1 h with a 1/5000 dilution of the secondary antibody  in TBS-T. Membranes were washed three times with TBS-T and blots were visualized with ECL Plus Western Blotting Detection Reagents  as per manufacturer's instructions.Whole-cell lysates were prepared by pelleting 1 ml each of the