A Versatile ΦC31 Based Reporter System for Measuring AP-1 and Nrf2 Signaling in Drosophila and in Tissue Culture

This paper describes the construction and characterization of a system of transcriptional reporter genes for monitoring the activity of signaling pathways and gene regulation mechanisms in intact Drosophila, dissected tissues or cultured cells. Transgenic integration of the reporters into the Drosophila germline was performed in a site-directed manner, using ΦC31 integrase. This strategy avoids variable position effects and assures low base level activity and high signal responsiveness. Defined integration sites furthermore enable the experimenter to compare the activity of different reporters in one organism. The reporter constructs have a modular design to facilitate the combination of promoter elements (synthetic transcription factor binding sites or natural regulatory sequences), reporter genes (eGFP, or DsRed.T4), and genomic integration sites. The system was used to analyze and compare the activity and signal response profiles of two stress inducible transcription factors, AP-1 and Nrf2. To complement the transgenic reporter fly lines, tissue culture assays were developed in which the same synthetic ARE and TRE elements control the expression of firefly luciferase

A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases

A transgenic mouse model for monitoring oxidative stress

Oxidative stress conditions enhance the production of reactive oxygen species resulting from a variety of stimuli, and are associated with various human diseases, including neurodegenerative disorders, inflammation, and various cancers. Though such associations have been closely studied using animal models, there has been no in vivo system for monitoring oxidative stress. We have developed an oxidative stress indicator that is dually regulated by induction at the transcriptional level, and by protein stabilisation at the post-translational level in Keap1-Nrf2 pathway. In vitro, our indicator elicited an intense and specific signal to oxidative stress among various agents, in a Keap1-Nrf2-dependent manner. Moreover, the transgenic animal expressing the indicator exhibited significant signals upon oxidative stress. These results indicate the usefulness of our system as an indicator of oxidative stress both in vitro and in vivo

High frequency of CHD7 mutations in congenital hypogonadotropic hypogonadism

Congenital hypogonadotropic hypogonadism (CHH) is characterized by lack of normal pubertal development due to deficient gonadotropin-releasing hormone (GnRH) secretion or action, and is caused by genetic defects in several genes. Mutations in the CHD7 gene cause CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and development, Genital hypoplasia and Ear abnormalities), but have also been found in patients with isolated CHH. The aim of this study was to identify CHD7 mutations in patients with CHH. Fifty Portuguese patients with CHH were screened for mutations in the CHD7 gene by DNA sequencing. Eight (16%) patients had CHD7 rare sequence variants that consisted of six missense (p.Gly388Glu, p.His903Pro, p.Thr1082Ile, p.Val1452Leu, p.Asp1854Gly, and p.Arg2065His) and two synonymous (p.Ser559Ser, and p.Ala2785Ala) mutations. Five of these mutations have never been reported before. Three CHD7 mutations occurred in patients that had mutations in additional CHH-genes. This study uncovered novel genetic variants that expand the known spectrum of mutations associated with CHH. The frequency of CHD7 mutations in this cohort was higher than that of other major CHH-genes and confirms the importance of including CHD7 in the genetic testing of CHH, even in the absence of additional CHARGE features.info:eu-repo/semantics/publishedVersio

Effects of an amylopectin and chromium complex on the anabolic response to a suboptimal dose of whey protein

Background Previous research has demonstrated the permissive effect of insulin on muscle protein kinetics, and the enhanced insulin sensitizing effect of chromium. In the presence of adequate whole protein and/or essential amino acids (EAA), insulin has a stimulatory effect on muscle protein synthesis, whereas in conditions of lower blood EAA concentrations, insulin has an inhibitory effect on protein breakdown. In this study, we determined the effect of an amylopectin/chromium (ACr) complex on changes in plasma concentrations of EAA, insulin, glucose, and the fractional rate of muscle protein synthesis (FSR). Methods Using a double-blind, cross-over design, ten subjects (six men, four women) consumed 6 g whey protein + 2 g of the amylopectin-chromium complex (WPACr) or 6 g whey protein (WP) after an overnight fast. FSR was measured using a primed, continuous infusion of ring-d5-phenylalanine with serial muscle biopsies performed at 2, 4, and 8 h. Plasma EAA and insulin were assayed by ion-exchange chromatography and ELISA, respectively. After the biopsy at 4 h, subjects ingested their respective supplement, completed eight sets of bilateral isotonic leg extensions at 80% of their estimated 1-RM, and a final biopsy was obtained 4 h later. Results Both trials increased EAA similarly, with peak levels noted 30 min after ingestion. Insulin tended (p = 0.09) to be higher in the WPACr trial. Paired samples t-tests using baseline and 4-h post-ingestion FSR data separately for each group revealed significant increases in the WPACr group (+0.0197%/h, p = 0.0004) and no difference in the WP group (+0.01215%/hr, p = 0.23). Independent t-tests confirmed significant (p = 0.045) differences in post-treatment FSR between trials. Conclusions These data indicate that the addition of ACr to a 6 g dose of whey protein (WPACr) increases the FSR response beyond what is seen with a suboptimal dose of whey protein alone

Genetic and Cellular Characterization of Caenorhabditis elegans Mutants Abnormal in the Regulation of Many Phase II Enzymes

Background: The phase II detoxification enzymes execute a major protective role against xenobiotics as well as endogenous toxicants. To understand how xenobiotics regulate phase II enzyme expression, acrylamide was selected as a model xenobiotic chemical, as it induces a large number and a variety of phase II enzymes, including numerous glutathione S-transferases (GSTs) in Caenorhabditis elegans. Methodology/Principal Findings: To begin dissecting genetically xenobiotics response pathways (xrep), 24 independent mutants of C. elegans that exhibited abnormal GST expression or regulation against acrylamide were isolated by screening about 3.5610 5 genomes of gst::gfp transgenic strains mutagenized with ethyl methanesulfonate (EMS). Complementation testing assigned the mutants to four different genes, named xrep-1,-2,-3, and-4. One of the genes, xrep-1, encodes WDR-23, a nematode homologue of WD repeat-containing protein WDR23. Loss-of-function mutations in xrep-1 mutants resulted in constitutive expression of many GSTs and other phase II enzymes in the absence of acrylamide, and the wild-type xrep-1 allele carried on a DNA construct successfully cured the mutant phenotype of the constitutive enzyme expression. Conclusions/Significance: Genetic and cellular characterization of xrep-1 mutants suggest that a large number of GSTs and other phase II enzymes induced by acrylamide are under negative regulation by XREP-1 (WDR-23), which is likely to be a functional equivalent of mammalian Keap1 and a regulator of SKN-1, a C. elegans analogue of cap-n-collar Nrf2 (nuclea

Congenital hypogonadotropic hypogonadism and constitutional delay of growth and puberty have distinct genetic architectures

Congenital hypogonadotropic hypogonadism (CHH) and constitutional delay of growth and puberty (CDGP) represent rare and common forms of GnRH deficiency, respectively. Both CDGP and CHH present with delayed puberty, and the distinction between these two entities during early adolescence is challenging. More than 30 genes have been implicated in CHH, while the genetic basis of CDGP is poorly understood. We characterized and compared the genetic architectures of CHH and CDGP, to test the hypothesis of a shared genetic basis between these disorders. Exome sequencing data were used to identify rare variants in known genes in CHH ( <i>n</i>  = 116), CDGP ( <i>n</i>  = 72) and control cohorts ( <i>n</i>  = 36 874 ExAC and <i>n</i>  = 405 CoLaus). Mutations in at least one CHH gene were found in 51% of CHH probands, which is significantly higher than in CDGP (7%, <i>P</i>  = 7.6 × 10 <sup>-11</sup> ) or controls (18%, <i>P</i>  = 5.5 × 10 <sup>-12</sup> ). Similarly, oligogenicity (defined as mutations in more than one gene) was common in CHH patients (15%) relative to CDGP (1.4%, <i>P</i>  = 0.002) and controls (2%, <i>P</i>  = 6.4 × 10 <sup>-7</sup> ). Our data suggest that CDGP and CHH have distinct genetic profiles, and this finding may facilitate the differential diagnosis in patients presenting with delayed puberty

Induction of Cytoprotective Pathways Is Central to the Extension of Lifespan Conferred by Multiple Longevity Pathways

Many genetic and physiological treatments that extend lifespan also confer resistance to a variety of stressors, suggesting that cytoprotective mechanisms underpin the regulation of longevity. It has not been established, however, whether the induction of cytoprotective pathways is essential for lifespan extension or merely correlated. Using a panel of GFP-fused stress response genes, we identified the suites of cytoprotective pathways upregulated by 160 gene inactivations known to increase Caenorhabditis elegans longevity, including the mitochondrial UPR (hsp-6, hsp-60), the ER UPR (hsp-4), ROS response (sod-3, gst-4), and xenobiotic detoxification (gst-4). We then screened for other gene inactivations that disrupt the induction of these responses by xenobiotic or genetic triggers, identifying 29 gene inactivations required for cytoprotective gene expression. If cytoprotective responses contribute directly to lifespan extension, inactivation of these genes would be expected to compromise the extension of lifespan conferred by decreased insulin/IGF-1 signaling, caloric restriction, or the inhibition of mitochondrial function. We find that inactivation of 25 of 29 cytoprotection-regulatory genes shortens the extension of longevity normally induced by decreased insulin/IGF-1 signaling, disruption of mitochondrial function, or caloric restriction, without disrupting normal longevity nearly as dramatically. These data demonstrate that induction of cytoprotective pathways is central to longevity extension and identify a large set of new genetic components of the pathways that detect cellular damage and couple that detection to downstream cytoprotective effectors.National Institute on Aging (AG16636

The Putative bZIP Transcripton Factor BzpN Slows Proliferation and Functions in the Regulation of Cell Density by Autocrine Signals in Dictyostelium

The secreted proteins AprA and CfaD function as autocrine signals that inhibit cell proliferation in Dictyostelium discoideum, thereby regulating cell numbers by a negative feedback mechanism. We report here that the putative basic leucine zipper transcription factor BzpN plays a role in the inhibition of proliferation by AprA and CfaD. Cells lacking BzpN proliferate more rapidly than wild-type cells but do not reach a higher stationary density. Recombinant AprA inhibits wild-type cell proliferation but does not inhibit the proliferation of cells lacking BzpN. Recombinant CfaD also inhibits wild-type cell proliferation, but promotes the proliferation of cells lacking BzpN. Overexpression of BzpN results in a reduced cell density at stationary phase, and this phenotype requires AprA, CfaD, and the kinase QkgA. Conditioned media from high-density cells stops the proliferation of wild-type but not bzpN− cells and induces a nuclear localization of a BzpN-GFP fusion protein, though this localization does not require AprA or CfaD. Together, the data suggest that BzpN is necessary for some but not all of the effects of AprA and CfaD, and that BzpN may function downstream of AprA and CfaD in a signal transduction pathway that inhibits proliferation