Jones AK et al. (1995) International C. elegans Meeting "CAN BLI-4 COMPLEMENT S.CEREVISIAE KEX2 MUTANTS?"

Thacker CM, Boone C, Jones AK, Rose AM

[International C. elegans Meeting, 1995]

The kex2/subtilisin proprotein convertase family of enzymes convert biologically inactive precursors into active secreted protein molecules. In C.elegans, the characterization of the bli-4 gene introduces a new member to the kex2/subtilisin family of endoproteases. bli-4 encodes at least four isoforms, of which the D isoform is structurally most similar to the KEX2 product in yeast. The trafficking signals of these gene products share significant sequence identity, providing an opportunity to learn about conservation of trafficking and localization signals between species. We are testing a cDNA clone of the blisterase D isoform for the ability to rescue an S.cerevisiae strain mutant in the homologous KEX2 gene. The cDNA clone has been constructed and inserted into a yeast expression vector. The resultant construct was transfected into a KEX2-deficient yeast strain. Transformants, produced on selective galactose media, were assayed for expression of blisterase D. However, preliminary experiments failed to indicate expression of the blisterase D clone. This may be a result of lack of expression of the bli-4 gene, or the blisterase product may not be appropriately folded or transported. Chimeric constructs may help to investigate the critical domain for complementation of the KEX2-deficient yeast. Further experimental approaches will include replacing the KEX2 protease domain with Bli-4, as well as the carboxy terminus, and investigating whether the KEX2 function can be complemented. If successful, this approach can also be used to generate more mutations in bli-4, in particular temperature sensitive mutants, since none have thus far been generated by direct C.elegans mutagenesis. Supported by the MRC of Canada.

Jones AK et al. (2005) Nat Rev Drug Discov "Chemistry-to-gene screens in Caenorhabditis elegans."

Jones AK, Sattelle DB, Buckingham SD

[Nat Rev Drug Discov, 2005]

The nematode worm Caenorhabditis elegans is a genetic model organism linked to an impressive portfolio of fundamental discoveries in biology. This free-living nematode, which can be easily and inexpensively grown in the laboratory, is also a natural vehicle for screening for drugs that are active against nematode parasites. Here, we show that chemistry-to-gene screens using this animal model can define targets of antiparasitic drugs, identify novel candidate drug targets and contribute to the discovery of new drugs for treating human diseases.

Jones AK et al. (1995) Worm Breeder's Gazette "Can bli-4 Complement S. cerevisiae kex2 Mutants?"

Jones AK, Boone C, Thacker CM, Rose AM

[Worm Breeder's Gazette, 1995]

Can bli-4 Complement S.cerevisiae kex2 Mutants?

Jones AK et al. (2004) Bioessays "Functional genomics of the nicotinic acetylcholine receptor gene family of the nematode, Caenorhabditis elegans."

Sattelle DB, Jones AK

[Bioessays, 2004]

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that bring about a diversity of fast synaptic actions. Analysis of the Caenorhabditis elegans genome has revealed one of the most-extensive and diverse nAChR gene families known, consisting of at least 27 subunits. Striking variation with possible functional implications has been observed in normally conserved motifs at the acetylcholine-binding site and in the channel-lining region. Some nAChR subunits are particular to neurons whilst others are present in both neurons and muscles. The localization of subunits in non-synaptic regions suggests novel roles for nAChRs. Genetic and heterologous expression studies have identified a subset of nAChR subunits that are important drug targets while the study of mutants has identified genes functionally-linked to nAChRs. Future studies using C. elegans offer the prospect of increasing our understanding of the functional diversity of a complex nAChR gene family as well as addressing the role of nAChRs and associated proteins in human disorders.

Jones AK et al. (2008) Invert Neurosci "The cys-loop ligand-gated ion channel gene superfamily of the nematode, Caenorhabditis elegans."

Jones AK, Sattelle DB

[Invert Neurosci, 2008]

The nematode, Caenorhabditis elegans, possesses the most extensive known superfamily of cys-loop ligand-gated ion channels (cys-loop LGICs) consisting of 102 subunit-encoding genes. Less than half of these genes have been functionally characterised which include cation-permeable channels gated by acetylcholine (ACh) and gamma-aminobutyric acid (GABA) as well as anion-selective channels gated by ACh, GABA, glutamate and serotonin. Following the guidelines set for genetic nomenclature for C. elegans, we have designated unnamed subunits as lgc genes (ligand-gated ion channels of the cys-loop superfamily). Phylogenetic analysis shows that several of these lgc subunits form distinct groups which may represent novel cys-loop LGIC subtypes.

Jones AK et al. (2007) Invert Neurosci "The nicotinic acetylcholine receptor gene family of the nematode Caenorhabditis elegans: an update on nomenclature."

Jones AK, Davis P, Hodgkin J, Sattelle DB

[Invert Neurosci, 2007]

The simple nematode, Caenorhabditis elegans, possesses the most extensive known gene family of nicotinic acetylcholine receptor (nAChR)-like subunits. Whilst all show greatest similarity with nAChR subunits of both invertebrates and vertebrates, phylogenetic analysis suggests that just over half of these (32) may represent other members of the cys-loop ligand-gated ion channel superfamily. We have introduced a novel nomenclature system for these "Orphan" subunits, designating them as lgc genes (ligand-gated ion channels of the cys-loop superfamily), which can also be applied in future to unnamed and uncharacterised members of the cys-loop ligand-gated ion channel superfamily. We present here the resulting updated version of the C. elegans nAChR gene family and related ligand-gated ion channel genes.

Jones AK et al. (2011) J Biol Chem "A Cys-loop mutation in the Caenorhabditis elegans nicotinic receptor subunit UNC-63 impairs but does not abolish channel function."

Maynard TP, Buckingham SD, Jones AK, Sattelle DB, Hernando G, Rayes D, Bouzat CB, Jones R, Al-Diwani A

[J Biol Chem, 2011]

The nematode Caenorhabditis elegans is an established model organism for studying neurobiology. UNC-63 is a C. elegans nicotinic acetylcholine receptor (nAChR) -subunit. It is an essential component of the levamisole-sensitive muscle nAChR (L-nAChR) and therefore plays an important role in cholinergic transmission at the nematode neuromuscular junction. Here, we show that worms with the unc-63(x26) allele, with its C151Y mutation disrupting the Cys-loop, have deficient muscle function reflected by impaired swimming (thrashing). Single-channel recordings from cultured muscle cells from the mutant strain showed a 100-fold reduced frequency of opening events and shorter channel openings of L-nAChRs compared with those of wild-type worms. Anti-UNC-63 antibody staining in both cultured adult muscle and embryonic cells showed that L-nAChRs were expressed at similar levels in the mutant and wild-type cells, suggesting that the functional changes in the receptor, rather than changes in expression, are the predominant effect of the mutation. The kinetic changes mimic those reported in patients with fast-channel congenital myasthenic syndromes. We show that pyridostigmine bromide and 3,4-diaminopyridine, which are drugs used to treat fast-channel congenital myasthenic syndromes, partially rescued the motility defect seen in unc-63(x26). The C. elegans unc-63(x26) mutant may therefore offer a useful model to assist in the development of therapies for syndromes produced by altered function of human nAChRs.

AK Jones et al. (2009) European Worm Neurobiology Meeting "A mutation in the Caenorhabditis elegans nicotinic acetylcholine receptor a subunit, unc-63, provides a model for fast channel congenital myasthenia"

C Bouzat, AK Jones, A Al-Diwani, TPR Maynard, DB Sattelle, G Hernando, D Rayes, SD Buckingham, R Jones

[European Worm Neurobiology Meeting, 2009]

Authors acknowledge support from MRC and OXION. Abstract: The nematode Caenorhabditis elegans is an established model organism for studying neurobiology since many synaptic molecular components are conserved both in the worm and humans. The unc-63 a subunit of the C. elegans nicotinic acetylcholine receptor (nAChR) plays an important role in fast cholinergic synaptic transmission at the nematode neuromuscular junction (1). Here we show that worms with the unc-63(x26) allele, with its .C151Y mutation that disrupts the Cys-loop, have deficient muscle function as displayed by impaired thrashing locomotion compared to wild-type worms (2). Single-channel recordings from muscle cells of the mutant strain show reduced number of active patches, a 100-fold reduced frequency of opening events, and abnormally reduced duration of channel openings similar to that observed in patients with fast-channel congenital myasthenia syndromes (FCCMS). Interestingly, a mutation in the equivalent position of the e subunit of the human muscle nAChR (.C128S) is associated with a FCCMS due to nAChR deficiency at the endplate (3). Therefore, disruption of the Cys-loop results in qualitatively similar actions on both nematode and human receptors. Therapy for FCCMS is limited in variety and success with 3,4-diaminopyridine (3,4-DAP) and anticholinesterase drugs such as pyridostigmine bromide (PB). We show that PB and 3,4-DAP can partially rescue the motility defect seen in unc-63(x26). Conclusions: We describe the first candidate C. elegans model for screening for drugs aimed at ameliorating the consequences of mutations in nAChRs associated with FCCMS. References: (1) Culetto E et al. J Biol Chem. 2004. 279:42476-83. (2) Jones AK et al. Hum Mol Genet. Submitted 2009. (3) Milone M et al. Ann N Y Acad Sci. 1998. 841:184-8.

Umair S et al. (2013) Exp Parasitol "Molecular and biochemical characterisation of arginine kinases in Haemonchus contortus and Teladorsagia circumcincta."

Bland RJ, Umair S, Simpson HV, Knight JS

[Exp Parasitol, 2013]

Full length cDNA encoding arginine kinases (AK) were cloned from Teladorsagia circumcincta (TcAK) and Haemonchus contortus (HcAK). The TcAK and HcAK cDNA (1080 bp) encoded 360 amino acid proteins. The predicted amino acid sequence showed 99% similarity with each other and 94% with a Caenorhabditis elegans AK. Soluble N-terminal His-tagged AK proteins were expressed in Escherichia coli strain BL21, purified and characterised. All binding sites were completely conserved in both proteins. The recombinant TcAK and HcAK had very similar kinetic properties: K(m) arginine was 0.35 mM, K(m) ATP was 0.8-0.9 mM and the pH optima were pH 7.5. Arginine analogues strongly inhibited recombinant enzyme activities (up to 80%), whilst other amino acids decreased activities by a maximum of 20%. TcAK and HcAK are potential vaccine candidates because of the strong antigenicity of invertebrate phosphagens and kinases and presence in metabolically active parts of the worm.

Zhai R et al. (2006) FEBS Lett "A novel nuclear-localized protein with special adenylate kinase properties from Caenorhabditis elegans."

Zhang G, Liu B, Zhai R, Meng G, Zheng X, Zhao Y

[FEBS Lett, 2006]

The adrenal gland protein AD-004 like protein (ADLP) from Caenorhabditis elegans was cloned and expressed in Escherichia coli. Enzyme assays showed that ADLP has special adenylate kinase (AK) properties, with ATP and dATP as the preferred phosphate donors. In contrast to all other AK isoforms, AMP and dAMP were the preferred substrates of ADLP; CMP, TMP and shikimate acid were also good substrates. Subcellular localization studies showed a predominant nuclear localization for this protein, which is different from AK1-AK5, but similar to that of human AK6. These results suggest that ADLP is more likely a member of the AK6 family. Furthermore, RNAi experiments targeting ADLP were conducted and showed that RNAi treatment resulted in the suppression of worm growth.