Reversible inhibitors of regulators of G-protein signaling identified in a high-throughput cell-based calcium signaling assay

Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric G-protein α subunits. RGS4 is enriched in the CNS and is proposed as a therapeutic target for treatment of neuropathological states including epilepsy and Parkinson's disease. Therefore, identification of novel RGS4 inhibitors is of interest. An HEK293-FlpIn cell-line stably expressing M₃-muscarinic receptor with doxycycline-regulated RGS4 expression was employed to identify compounds that inhibit RGS4-mediated suppression of M₃-muscarinic receptor signaling. Over 300,000 compounds were screened for an ability to enhance Gα_q-mediated calcium signaling in the presence of RGS4. Compounds that modulated the calcium response in a counter-screen in the absence of RGS4 were not pursued. Of the 1365 RGS4-dependent primary screen hits, thirteen compounds directly target the RGS-G-protein interaction in purified systems. All thirteen compounds lose activity against an RGS4 mutant lacking cysteines, indicating that covalent modification of free thiol groups on RGS4 is a common mechanism. Four compounds produce > 85% inhibition of RGS4-G-protein binding at 100 μM, yet are > 50% reversible within a ten-minute time frame. The four reversible compounds significantly alter the thermal melting temperature of RGS4, but not G-protein, indicating that inhibition is occurring through interaction with the RGS protein. The HEK cell-line employed for this study provides a powerful tool for efficiently identifying RGS-specific modulators within the context of a GPCR signaling pathway. As a result, several new reversible, cell-active RGS4 inhibitors have been identified for use in future biological studies.     

Chromosomal assignment of porcine pregnancy-associated glycoprotein gene family

This study presents the chromosomal assignment of a multiple pregnancy-associated glycoprotein (PAG) gene family in the domestic pig (pPAG). The pPAG locus was identified by physical mapping (fluorescent in situ hybridisation—FISH; with various probes), and additionally confirmed by Southern hybridisation of pPAG amplicons using laser microdissected Sus scrofa chromosome 1 (SSC1), as genomic templates. Various pPAG probes were produced with the use of diverse identified templates: pPAG1-6, -8, -10 cDNAs (GenBank: L34360–1, AF315377, AF272734, AY188554, AF272735, AY373029 and AY775784, respectively), or genomic DNA (gDNA) probes of pPAG2 gene and its promoter (GenBank: U39198–9, U39762–3, U41421–4). All probes, including long gDNA probes (～9.2 kbp GpPAG2 gene; ～2.8 kbp GpPAG2 promoter), a shorter cDNA probe (PlpPAG4, 1385 bp) and amplified pPAG2-like probes (ApPAG2L) specific for cDNA inserts of pPAG2-like gene subfamily (pPAG2, -4, -6, -8 and -10; 1283–1385 bp) were produced by random priming using biotin-labelled deoxynucleotides (16-dUTP). Numerous FISH mappings with various pPAG probes revealed the chromosomal assignment of the pPAG gene family to the long arm of porcine chromosome 1 (SSC1q16–q24 region). This cytogenetic assignment was confirmed by Southern hybridisation (with ³²P-labelled pPAG10 probe) of multiple distinct pPAG amplicons (603–3943 bp) produced with the use of 25 laser microdissected SSC1, as gDNA templates. This is the first study identifying the chromosomal locus of the pPAG gene family in the pig.     

Characterization of the human NIPSNAP1 gene from 22q12: a member of a novel gene family

Rapid progress in sequencing of human and other genomes allows high-resolution analysis of their gene content on the basis of comparison between species. We have used a combined computer and biochemical approach to characterize 135 kb of human genomic sequence from 22q12 and discovered a new 10 exon gene, termed NIPSNAP1, located between the neurofibromatosis type 2 and the pK1.3 genes. The NIPSNAP1 gene spans 26 kb of genomic sequence and shows two large introns in the 5′-region. All exon–intron junctions contain the gt/ag consensus splice site. The putative promoter of the NIPSNAP1 gene is TATA-less and resides in a GC-rich island characteristic of housekeeping genes. The NIPSNAP1 mRNA is 2.1 kb, is expressed ubiquitously at variable levels, with the highest expression in liver, is terminated by an uncommon ATTAAA polyadenylation site, and is capable of encoding a 284-amino-acid protein. This NIPSNAP1 protein has a strong sequence similarity limited to the central portion of a hypothetical protein (acc. P34492) from chromosome III of C. elegans, in which the other portions resemble a 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein. Thus, the NIPSNAP1 gene is a member of an evolutionarily well conserved, novel gene family with two members in human and mouse that have now been characterized, and one member in C. elegans. The second human gene, NIPSNAP2, is localized in the vicinity of marker D7S499 on chromosome 7. Although the function of the NIPSNAP protein family is unknown, clues about its role may reside in the co-expression of the C. elegans orthologue, within an operon encoding protein motifs known to be involved in vesicular transport.     

Localization and genomic organization of sheep antimicrobial peptide genes

Antimicrobial peptides are an abundant and diverse component of animal innate immunity. Within mammalian species, defensins and cathelicidins are the two principal antimicrobial peptide families. We identified and sequenced ten new sheep genes which encode potential antimicrobial peptides including two β-defensins and eight cathelicidins. We mapped the two-exon β-defensin genes to sheep chromosome 26 and the four-exon cathelicidin genes to sheep chromosome 19 using sheep–hamster somatic cell hybrids in conjunction with flow-sorted sheep chromosomes. These assignments confirm homology between sheep, cattle, mouse, and human antimicrobial peptide gene families. Contig construction for the sheep cathelicidin gene family demonstrates that three genes, OaDodeA, OaDodeB, and OaMAP-34, are present head-to-tail in a 14.5 kb region, and that four proline/arginine-rich genes, OaBac5, OaBac7.5, OaBac11, and OaBac6, are arranged head-to-tail in a region covering 30.5 kb. This richly diverse family of sheep cathelicidin peptides is encoded in a gene array which may reflect the mechanism of its evolution.     

Structure and chromosome localization of the human CASP8 gene

The human CASP8 gene, whose product is also known as caspase 8 and FLICE, encodes an interleukin-1β converting enzyme (ICE)-related cysteine protease that is activated by the engagement of several different death receptors. Caspase 8 is immediately recruited to the Fas receptor once it oligomerizes, and its protease activity is crucial for the apoptotic response generated by the resulting death-inducing signaling complex (DISC). We report here that the CASP8 gene contains at least 11 exons spanning ∼30 kb on human chromosome band 2q33–34. This region of human chromosome 2 was previously reported as the location of the CASP10 gene, whose product is closely related to caspase 8. Chromosome 2 band q33–34 is also involved in tumorigenesis, with loss of heterogeneity (LOH) being reported in a number of tumors. We also report EcoRI and HindIII polymorphisms that may prove to be useful in disease analysis. Both caspases 8 and 10 contain long pro-domains with duplicated death effector domains (DEDs), as well as their corresponding cysteine protease catalytic domains. Thus, it appears that CASP8 and CASP10 have evolved by tandem gene duplication, much like the CASP1, CASP4 and CASP5 gene cluster on human chromosome 11q22.2–22.3.     

Frigoriglobus tundricola gen. nov., sp. nov., a psychrotolerant cellulolytic planctomycete of the family Gemmataceae from a littoral tundra wetland

Planctomycetes of the family Gemmataceae are characterized by large genome sizes and cosmopolitan distribution in freshwater and terrestrial environments but their ecological functions remain poorly understood. In this study, we characterized a novel representative of this family, strain PL17^T, which was isolated from a littoral tundra wetland and was capable of growth on xylan and cellulose. Cells of this isolate were represented by pink-pigmented spheres that multiplied by budding and occurred singly or in short chains and aggregates. Strain PL17^T was obligately aerobic, mildly acidophilic chemoorganotrophic bacterium, which displayed good tolerance of low temperatures. The major fatty acids were C_18:0, C_16:1ω5, and βOH-C_16:1; the major polar lipid was trimethylornithine. The genome of strain PL17^T consisted of a 9.83 Mb chromosome and a 24.69 kb plasmid. The G + C contents of the chromosomal and plasmid DNA were 67.4 and 62.3 mol%, respectively. Over 8900 potential protein-coding genes were identified in the genome including a putative cellulase that contains a domain from the GH5 family of glycoside hydrolases. The genome of strain PL17^T contained one linked and one unlinked rRNA operons with 16S rRNA gene sequences displaying 94.5% similarity to that in Gemmata obscuriglobus UQM2246^T. Based on the results of comparative phenotypic, chemotaxonomic and phylogenomic analyses, we propose to classify strain PL17^T (= CECT 9407^T = VKM B-3467^T) as representing a novel genus and species of the family Gemmataceae, Frigoriglobus tundricola gen. nov., sp. nov.     

Novel gene variants predict serum levels of the cytokines IL-18 and IL-1ra in older adults

Activation of inflammatory pathways measured by serum inflammatory markers such as interleukin-18 (IL-18) and interleukin-1 receptor antagonist (IL-1ra) is strongly associated with the progression of chronic disease states in older adults. Given that these serum cytokine levels are in part a heritable trait, genetic variation may predict increased serum levels. Using the Cardiovascular Health Study and InCHIANTI cohorts, a genome-wide association study was performed to identify genetic variants that influence IL-18 and IL-1ra serum levels among older adults. Multiple linear regression models characterized the association between each SNP and log-transformed cytokine values. Tests for multiple independent signals within statistically significant loci were performed using haplotype analysis and regression models conditional on lead SNP in each region. Multiple SNPs were associated with these cytokines with genome-wide significance, including SNPs in the IL-18-BCO gene region of chromosome 2 for IL-18 (top SNP rs2250417, P = 1.9 × 10^–32) and in the IL-1 gene family region of chromosome 2 for IL-1ra (rs6743376, P = 2.3 × 10^–26). Haplotype tests and conditional linear regression models showed evidence of multiple independent signals in these regions. Serum IL-18 levels were also associated with a region on chromosome 2 containing the NLRC4 gene (rs12989936, P = 2.7 × 10^–19). These data characterize multiple robust genetic signals that influence IL-18 and IL-1ra cytokine production. In particular, the signal for serum IL-18 located on chromosome two is novel and potentially important in inflammasome triggered chronic activation of inflammation in older adults. Replication in independent cohorts is an important next step, as well as molecular studies to better understand the role of NLRC4.     

amlC,Another amylolytic gene maps close to the amlB locus in Streptomyces lividans TK24

The region located upstream of the α-amylase gene (amlB) of Streptomyces lividans TK24 (Yin et al., 1997) contains a 2978-bp-long ORF divergent from amlB, and designated amlC. amlC Encodes a 993 amino acid (aa) protein with a calculated molecular weight of 107.054 kDa. On the basis of sequence similarity as well as enzymatic activity, AmlC is likely to belong to the 1,4-α-d-glucan glucanohydrolase family. amlC is transcribed as a unique 3 kb leaderless monocistronic mRNA. Primer extension experiments allowed the identification of promoter sequences that do not resemble the typical eubacterial promoter sequences. amlC was successfully disrupted and was mapped at approx. 700 kb from a chromosomal end of S. lividans TK24, 100 kb on the right of the amplifiable unit AUD1 (Volff et al., 1996). Nevertheless, amlC disruption seemed to be accompanied by extensive rearrangements of the 2500-kb DraI–II fragment of the chromosome.     

R4 regulators of G protein signaling (RGS) identify an ancient MHC-linked synteny group

Regulators of G protein signaling (RGS) are key regulators of G protein signaling. RGS proteins of the R4 RGS group are composed of a mere RGS domain and are mainly involved in immune response modulation. In both human and mouse, most genes encoding the R4 RGS proteins are located in the same region of chromosome 1. We show here that the RGS1/RGS16 neighborhood constitutes a synteny group well conserved across tetrapods and closely linked to the MHC paralogon of chromosome 1. Genes located in the RGS1/RGS16 region have paralogs close to the MHC on chromosome 6 or close to the other MHC paralogons. In amphioxus, a cephalochordate, these genes possess orthologs that are located in the same scaffolds as a number of markers defining the proto-MHC in this species (Abi-Rached et al., Nat Genet 31:100–115, 2002). We therefore propose that the RGS1/RGS16 region provides useful markers to investigate the origins and the evolution of the MHC. In addition, we show that some genes of the region appear to have immune functions not only in human, but also in Xenopus.     

The 1.6 Mb chromosome carrying the avirulence gene AvrPik in Magnaporthe oryzae isolate 84R-62B is a chimera containing chromosome 1 sequences

A genetic map was constructed previously from a cross between Magnaporthe oryzae isolates 84R-62B and Y93-245c-2, and genetic markers closely linked to the cultivar-specific avirulence (Avr) gene, AvrPik, were assigned to a 1.6 Mb small chromosome of 84R-62B that is absent from Y93-245c-2. In the present study, the 1.6 Mb chromosome was characterized by using contour-clamped homogeneous electric fields (CHEF) electrophoresis and hybridization analysis. CHEF electrophoresis analysis showed that the 1.6 Mb chromosome was inherited in Mendelian fashion, and co-segregated with AvrPik. Southern hybridization analysis revealed that the 1.6 Mb chromosome carried sequences only distributed to the supernumerary chromosome in M. oryzae isolates, as well as sequences corresponding to those in the supercontig 17 of chromosome 1 in the M. grisea database. Thus, we conclude that the Mendelian 1.6 Mb chromosome is a chimera containing sequences from chromosome 1 and from supernumerary chromosomes in M. oryzae.     

Spraguea lophii (Microsporidia) parasite of the teleost fish,Lophius piscatorius from Tunisian coasts: Evidence for an extensive chromosome length polymorphism

A microsporidian of the genus Spraguea was found parasitizing the nervous tissues of Lophius piscatorius collected from various localities in the Mediterranean coastal areas of Tunisia. The tissue localization, the infection focus aspect and sporal dimorphism are characteristics of Spraguea lophii species. Molecular data based on partial sequence of SSUrRNA encoding gene shows few nucleotide polymorphisms, compared to all described Spraguea isolates. Molecular karyotype obtained on pulsed field gel electrophoresis (1D-PFGE) shows a profile with 14 stained bands in the range of 230–880 kbp and a genome size estimated to 6.700 kbp. The rare cutter endonuclease MluI KARD 2-D-PFGE fingerprint shows an extensive chromosome length polymorphism, but the number of chromosome is unchanged and consists of 15 different molecules. The extensive chromosome length polymorphism is associated to a reduced number of genetic events.     

Molecular and biochemical characterizations of a novel arthropod endo-β-1,3-glucanase from the Antarctic springtail,Cryptopygus antarcticus,horizontally acquired from bacteria

Collembolan species have been known to have β-1,3-glucanase activity and yet the genes coding such enzymes have not been demonstrated. We report here a novel arthropod endo-β-1,3-glucanase gene CaLam from the Antarctic springtail, Cryptopygus antarcticus. The open reading frame consists of 813 bp encoding 270 amino acids with a putative signal peptide and a typical motif of glycosyl hydrolase family 16 (GHF16), E–I–D–I–T–E. The recombinant protein expressed in E. coli shows the hydrolytic activity toward laminarin (K_m ～ 9.98 mg/mL) with an optimal temperature 50 °C and an optimal pH 6.0. CaLam digests laminarin and laminarioligosaccharides except laminaribiose as an endo-β-1,3-glucanase, releasing glucose, laminaribiose and laminaritriose as the major products. Analyses of molecular phylogeny of CaLam and its protein structure reveal that CaLam is closely related with bacterial β-1,3-glucanases more than with the eukaryotic homologues. Even so, the genomic structure of the CaLam gene consisting of six exons interspersed with approximately 57 to 63 bp introns confirms that it is endogenous in the genome of the Antarctic springtail. These results suggest that CaLam should have been transferred from bacteria to the lineage of the Collembolan species by horizontal gene transfer.     

Kinetic characterization of human phosphopantothenoylcysteine synthetase

Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the formation of phosphopantothenoylcysteine from (R)-phosphopantothenate and l-cysteine with the concomitant consumption of a nucleotide triphosphate. Herein, the human coaB gene encoding PPCS is cloned into pET23a and overexpressed in E. coli BL21(DE3), to yield 10 mg of purified enzyme per liter of culture. Detailed kinetic studies found that this PPCS follows a similar Bi Uni Uni Bi Ping Pong mechanism as previously described for the E. faecalis PPCS, except that the human enzyme can use both ATP and CTP with similar affinity. One significant difference for human PPCS catalysis with respect to ATP and CTP is that the enzyme shows cooperative binding of ATP, measured as a Hill constant of 1.7. PPCS catalysis under CTP conditions displayed Michaelis constants of 265 μM, 57 μM, and 16 μM for CTP, PPA, and cysteine, respectively, with a k_cat of 0.53 ± 0.01 s^? 1 for the reaction. Taking into account the cooperativity under ATP condition, PPCS exhibited Michaelis constants of 269 μM, 13 μM, and 14 μM for ATP, PPA, and cysteine, respectively, with a k_cat of 0.56 s^? 1 for the reaction. Oxygen transfer studies found that ¹⁸O from [carboxyl-¹⁸O] phosphopantothenate is incorporated into the AMP or CMP produced during PPCS catalysis, consistent with the formation of a phosphopantothenoyl cytidylate or phosphopantothenoyl adenylate intermediate, supporting similar catalytic mechanisms under both CTP and ATP conditions. Inhibition studies with GTP and UTP as well as product inhibition studies with CMP and AMP suggest that human PPCS lacks strong nucleotide selectivity.     

Nonsense mutations in CABC1/ADCK3 cause progressive cerebellar ataxia and atrophy

Hereditary ataxias are genetic disorders characterized by uncoordinated gait and often poor coordination of hands, speech, and eye movements. Frequently, atrophy of the cerebellum occurs. Many ataxias are autosomal dominant, but autosomal recessive (AR) disease occurs as well. Homozygosity mapping in a consanguineous family with three affected children with progressive cerebellar ataxia and atrophy revealed a candidate locus on chromosome 1, containing the CABC1/ADCK3 (the chaperone, ABC1 activity of bc1 complex homologue) gene. CABC1/ADCK3 is the homologue of the yeast Coq8 gene, which is involved in the ubiquinone biosynthesis pathway. Mutation analysis of this gene showed a homozygous nonsense mutation (c.1042C > T, p.R348X). Eight additional patients with AR cerebellar ataxia and atrophy were screened for mutations in the CABC1/ADCK3 gene. One patient was compound heterozygous for the same c.1042C > T mutation and a second nonsense mutation (c.1136T > A, p.L379X). Both mutations created a premature stop codon, triggering nonsense mediated mRNA decay as the pathogenic mechanism. We found no evidence of a Dutch founder for the c.1042C > T mutation in AR ataxia. We report here the first nonsense mutations in CABC1 that most likely lead to complete absence of a functional CABC1 protein. Our results indicate that CABC1 is an important candidate for mutation analysis in progressive cerebellar ataxia and atrophy on MRI to identify those patients, who may benefit from CoQ10 treatment.     

Identification of a five-gene signature of the RGS gene family with prognostic value in ovarian cancer

The RGS (regulator of G protein signaling) gene family, which includes negative regulators of G protein-coupled receptors, comprises important drug targets for malignant tumors. It is thus of great significance to explore the value of RGS family genes for diagnostic and prognostic prediction in ovarian cancer. The RNA-seq, immunophenotype, and stem cell index data of pan-cancer, The Cancer Genome Atlas (TCGA) data, and GTEx data of ovarian cancer were downloaded from the UCSC Xena database. In the pan-cancer database, the expression level of RGS1, RGS18, RGS19, and RGS13 was positively correlated with stromal and immune cell scores. Cancer patients with high RGS18 expression were more sensitive to cyclophosphamide and nelarabine, whereas those with high RGS19 expression were more sensitive to cladribine and nelarabine. The relationship between RGS family gene expression and overall survival (OS) and progression-free survival (PFS) of ovarian cancer patients was analyzed using the KM-plotter database, RGS17, RGS16, RGS1, and RGS8 could be used as diagnostic biomarkers of the immune subtype of ovarian cancer, and RGS10 and RGS16 could be used as biomarkers to predict the clinical stage of this disease. Further, Lasso cox analysis identified a five-gene risk score (RGS11, RGS10, RGS13, RGS4, and RGS3). Multivariate COX analysis showed that the risk score was an independent prognostic factor for patients with ovarian cancer. Immunohistochemistry and the HPA protein database confirmed that the five-gene signature is overexpressed in ovarian cancer. GSEA showed that it is mainly involved in the ECM-receptor interaction, TGF-beta signaling pathway, Wnt signaling pathway, and chemokine signaling pathway, which promote the occurrence and development of ovarian cancer. The prediction model of ovarian cancer constructed using RGS family genes is of great significance for clinical decision making and the personalized treatment of patients with ovarian cancer.     

Structure and mapping of the human β-defensin HBD-2 gene and its expression at sites of inflammation

We cloned a second human β-defensin gene, HBD-2, and determined its gene structure and expression in inflamed tissue sections. The entire gene spanned about 2 kb with two small exons and one intron. Radiation hybrid studies confirmed the location on chromosome 8p, were consistent with the order HNP-1, HBD-1 and HBD-2, and located HBD-2 as the most centromeric of the genes. By three-color fluorescence in situ hybridization on both free chromatin fiber mapping and interphase mapping, HBD-1, HBD-2 and HNP-1 were mapped to chromosome 8p23. HBD-1 was within 40–100 kb of HNP-1, while HBD-2 was about 500–600 kb from HBD-1, with the most likely order HNP-1, HBD-1, HBD-2. The expression of HBD-2 was locally regulated by inflammation. HBD-2 mRNA was markedly increased in the epidermis surrounding inflamed regions, but not detectable in adjacent non-inflamed areas, a distribution that was confirmed at the peptide level by immunostaining with HBD-2 antibody. The HBD-2 gene is the first member of the human defensin family that is locally inducible by inflammation.