Roos MH et al. (1993) Pharmacol Ther "Molecular aspects of drug resistance in parasitic helminths."

Veenstra JG, Roos MH, Kooyman FN, Boersema JH, Kwa MSG

[Pharmacol Ther, 1993]

Parasitic helminths (worms) cause serious infectious diseases in humans and animals. As control of these infections relies mostly on chemotherapeutics, the anthelmintics, resistance has developed against most of these drugs in several parasite species. These resistant parasites are being used to elucidate the molecular mechanisms of drug action.

Simpson-Louredo L et al. (2019) Antonie Van Leeuwenhoek "Detection and virulence potential of a phospholipase D-negative Corynebacterium ulcerans from a concurrent diphtheria and infectious mononucleosis case."

Burkovski A, Santana MM, Antunes CA, Silva CMF, Mattos-Guaraldi AL, Hirata R, Villas Boas MHS, Hacker E, Souza NF, Nagao PE, Simpson-Louredo L

[Antonie Van Leeuwenhoek, 2019]

Diphtheria byCorynebacterium ulceransis increasingly occurring inchildren, adolescents and adults.In addition todiphtheria toxin(DT), phospholipase D (PLD) is considered a virulence factor ofC. ulcerans. In the present study,a first case ofconcurrent diphtheria bya PLD-negative C. ulceransand infectious mononucleosis (IM) was verified. Clinical and microbiological profiles and binding properties to human Fibrinogen (Fbg), Fibronectin (Fn) and type I collagen (col I) biotinylated proteins and virulence to Caenorhabditis elegans were investigated for C. ulcerans strain 2590 (clinical isolate) and two control strains, including PLD-positive BR-AD22 wild type and PLD-negative ELHA-1 PLD mutant strains. MALDI-TOF assays and a multiplex PCR of genes coding for potentially toxigenic corynebacteria identified strain 2590 as non-DT producing. Interestingly, strain 2590 did not express PLD activity in the CAMP test although the presence ofthe pldgene was verified. PLD-negative 2590 and a PLD-positive 210932 strains showed similar affinity to Fbg, Fn and type I collagen. C. eleganswere able to escape fromC. ulcerans strains, independent of PLD and DT production.Higher mortality of nematodes was verified for PLD-negative strains. Additional studies concerning multifactorial virulence potential of C. ulcerans, including environmental conditions remain necessary.

Greene D et al. (2008) Biophys J "Single-molecule force spectroscopy reveals a stepwise unfolding of Caenorhabditis elegans giant protein kinase domains."

Gernert KM, Benian GM, Sutton B, Garcia T, Greene D, Oberhauser AF

[Biophys J, 2008]

Myofibril assembly and disassembly are complex processes that regulate overall muscle mass. Titin kinase has been implicated as an initiating catalyst in signaling pathways that ultimately result in myofibril growth. In titin, the kinase domain is in an ideal position to sense mechanical strain that occurs during muscle activity. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and autoinhibitory/regulatory region. Molecular dynamics simulations suggest that human titin kinase acts as a force sensor. However, the precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Here we measured the mechanical properties of the kinase domain and flanking Ig/Fn domains of the C. elegans titin-like proteins twitchin and TTN-1 using single-molecule atomic force microscopy. Our results show that these kinase domains have significant mechanical resistance, unfolding at forces similar to those for Ig/Fn beta-sandwich domains (30-150pN). Further, our AFM data is consistent with molecular dynamic simulations, which show that these kinases unfold in a stepwise fashion, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data support the hypothesis that titin kinase may function as an effective force sensor.

Bullard B et al. (2002) J Muscle Res Cell Motil "Varieties of elastic protein in invertebrate muscles."

Leonard K, Linke WA, Bullard B

[J Muscle Res Cell Motil, 2002]

Elastic proteins in the muscles of a nematode (Caenorhabditis elegans), three insects (Drosophila melanogaster, Anopheles gambiae, Bombyx mori) and a crustacean (Procambus clarkii) were compared. The sequences of thick filament proteins, twitchin in the worm and projectin in the insects, have repeating modules with fibronectin-like (Fn) and immunoglobulin-like (Ig) domains conserved between species. Projectin has additional tandem Igs and an elastic PEVK domain near the N-terminus. All the species have a second elastic protein we have called SLS protein after the Drosophila gene, sallimus. SLS protein is in the I-band. The N-terminal region has the sequence of kettin which is a spliced product of the gene composed of Ig-linker modules binding to actin. Downstream of kettin, SLS protein has two PEVK domains, unique sequence, tandem Igs, and Fn domains at the end. PEVK domains have repeating sequences: some are long and highly conserved and would have varying elasticity appropriate to different muscles. Insect indirect flight muscle (IFM) has short I-bands and electron micrographs of Lethocerus IFM show fine filaments branching from the end of thick filaments to join thin filaments before they enter the Z-disc. Projectin and kettin are in this region and the contribution of these to the high passive stiffness of Drosophila IFM myofibrils was measured from the force response to length oscillations. Kettin is attached both to actin near the Z-disc and to the end of thick filaments, and extraction of actin or digestion of kettin leads to rapid decrease in stiffness; residual tension is attributable to projectin. The wormlike chain model for polymer elasticity fitted the force-extension curve of IFM myofibrils and the number of predicted Igs in the chain is consistent with the tandem Igs in Drosophila SLS protein. We conclude that passive tension is due to kettin and projectin, either separate or

Diaz-Balzac CA et al. (2015) Cell Rep "The Adhesion Molecule KAL-1/anosmin-1 Regulates Neurite Branching through a SAX-7/L1CAM-EGL-15/FGFR Receptor Complex."

Lazaro-Pena MI, Bulow HE, Ramos-Ortiz GA, Diaz-Balzac CA

[Cell Rep, 2015]

Neurite branching is essential for correct assembly of neural circuits, yet it remains a poorly understood process. For example, the neural cell adhesion molecule KAL-1/anosmin-1, which is mutated in Kallmann syndrome, regulates neurite branching through mechanisms largely unknown. Here, we show that KAL-1/anosmin-1 mediates neurite branching as an autocrine co-factor with EGL-17/FGF through a receptor complex consisting of the conserved cell adhesion molecule SAX-7/L1CAM and the fibroblast growth factor receptor EGL-15/FGFR. This protein complex, which appears conserved in humans, requires the immunoglobulin (Ig) domains of SAX-7/L1CAM and the FN(III) domains of KAL-1/anosmin-1 for formation in vitro as well as function in vivo. The kinase domain of the EGL-15/FGFR is required for branching, and genetic evidence suggests that ras-mediated signaling downstream of EGL-15/FGFR is necessary to effect branching. Our studies establish a molecular pathway that regulates neurite branching during development of the nervous system.

von Castelmur E et al. (2012) Proc Natl Acad Sci U S A "Identification of an N-terminal inhibitory extension as the primary mechanosensory regulator of twitchin kinase."

Strumpfer J, Benian GM, Qadota H, Mayans O, Bogomolovas J, Franke B, Konarev PV, Schulten K, von Castelmur E, Labeit S, Barbieri S, Svergun DI

[Proc Natl Acad Sci U S A, 2012]

Titin-like kinases are an important class of cytoskeletal kinases that intervene in the response of muscle to mechanical stimulation, being central to myofibril homeostasis and development. These kinases exist in autoinhibited states and, allegedly, become activated during muscle activity by the elastic unfolding of a C-terminal regulatory segment (CRD). However, this mechano-activation model remains controversial. Here we explore the structural, catalytic, and tensile properties of the multidomain kinase region of Caenorhabditis elegans twitchin (Fn(31)-Nlinker-kinase-CRD-Ig(26)) using X-ray crystallography, small angle X-ray scattering, molecular dynamics simulations, and catalytic assays. This work uncovers the existence of an inhibitory segment that flanks the kinase N-terminally (N-linker) and that acts synergistically with the canonical CRD tail to silence catalysis. The N-linker region has high mechanical lability and acts as the primary stretch-sensor in twitchin kinase, while the CRD is poorly responsive to pulling forces. This poor response suggests that the CRD is not a generic mechanosensor in this kinase family. Instead, the CRD is shown here to be permissive to catalysis and might protect the kinase active site against mechanical damage. Thus, we put forward a regulatory model where kinase inhibition results from the combined action of both N- and C-terminal tails, but only the N-terminal extension undergoes mechanical removal, thereby affording partial activation. Further, we compare invertebrate and vertebrate titin-like kinases and identify variations in the regulatory segments that suggest a mechanical speciation of these kinase classes.

Lewis JA et al. (1981) Worm Breeder's Gazette "first worm receptor found in New York City."

Lewis JA, Fleming JT

[Worm Breeder's Gazette, 1981]

Encouraged by Stretton & Co.'s result that many minutes are required to wash the muscle-contracting effect of meta-aminolevamisole out of Ascaris muscle strips, we've synthesized tritiated meta- aminolevamisole of high specific radioactivity. By thin layer chromatography, the drug is greater than 97% radiochemically pure, has the same high biological potency on cut C. onradioactive drug, and has a specific radioactivity of 15 to 30 Ci/mmole. We incubate the radioactive drug with total worm extract and collect any radioactivity bound to membrane fragments by passing the extract through a glass fiber fiIter. A control assay is done in the presence of a thousand fold excess of nonradioactive drug. Specific binding is the difference between the total binding (first assay) and nonspecific binding (second assay). We find that the wiId type nematode containing a high affinity, saturable binding activity. There is about 1 picomole of this activity per gram of worms, having a dissociation constant of roughly 1 x 10(-8) M. In agreement with Stretton's original finding, the drug dissociates very slowly from its bound state (half life ~ 10 min.). The formation of the bound state also occurs relatively slowly. The rate constant is ~ 1 x 10(5) liter/mole-sec at 0 C, several orders of magnitude slower than diffusion-limited binding, implying that a rate-limiting step such as a protein conformational change might be involved with the formation of the bound state observed fn vitro. We have screened extracts of several levamisole-resistant mutants having little or no response to the drug as living worms. ) al binding activity while 9) unc-29(x29)e. Several other mutants, 0),lev-7(x13), unc-29(e1O72)normally have activity altered in amount or affinity. Anyway, all this is a definite first of some sort. We'll try to figure out what and let you know more at the Worm Meeting.

Yu TW et al. (1998) West Coast Worm Meeting "Mechanisms of Cell and Growth Cone Guidance by Sax-3"

Prehoda K, Lim WA, Lee D, Yu TW, Bargmann CI

[West Coast Worm Meeting, 1998]

We are interested in the mechanisms that underlie cell and growth cone guidance in the development of the nervous system: what are the extracellular cues involved, and how is extracellular information translated into cytoskeletal changes and directed movement. Previous work from our lab has shown that sax-3 mutants exhibit a variety of defects in cell and axon migrations, including anterior misrouting of amphid axons, abnormal midline crossing of PVQ axons, wandering HSN axons and amphid neuron migration defects. sax-3 mutants are also variably Notch. sax-3 encodes a protein with immunoglobulin (Ig) and fibronectin (FN) type III extracellular motifs reminiscent of the neural cell adhesion molecule family, and a large cytoplasmic domain. It is highly similar to the Drosophila Roundabout guidance receptor and two mammalian genes. The cytoplasmic domain of sax-3 contains conserved proline-rich motifs that may couple this guidance cue to downstream effectors. To understand the mechanism of sax-3 guidance, we have used a candidate protein approach to identify proteins that interact with the sax-3 cytoplasmic domain in vitro. One such candidate, Mena, is a cytoskeletal protein implicated in the control of microfilament dynamics. We find that Mena specifically binds SAX-3. In addition, preliminary results show that RNA interference with a putative C. elegans homolog of Mena (C-ena) results in sax-3-like phenotypes in the F1 progeny, including uncoordination, cell migration defects and notched heads. The sax-3 cytoplasmic domain also appears to be a target for SH3 domain binding. An appealing candidate is the C. elegans protein ZK470.5, which we have isolated in a yeast two-hybrid screen. ZK470.5 encodes an adapter protein with three SH3 and two SH2 domains, and is closely related to vertebrate Nck (42% id) and Drosophila dreadlocks (40% id). In flies, dreadlocks is implicated in photoreceptor axon guidance and targetting. We therefore identify ZK470.5 as C. elegans dnr (dock/Nck-related). To complement these biochemical approaches we are also screening for suppressors of sax-3. We have identified over forty mutants that suppress amphid axon defects of a temperature-sensitive allele of sax-3 (ky200). We will report progress from these screens at the meeting.

Oates AC et al. (1995) Worm Breeder's Gazette "How many PTKs to organise a worm?"

Wilks AF, Oates AC

[Worm Breeder's Gazette, 1995]

Protein tyrosine kinases (PTKs) play important roles in coordinating metazoan cell behavior, although only one such example, let-23, is understood clearly in the nematode worm. Therefore we have undertaken a screen for PTK-related sequences from the C. elegans genome using PCR and PTK specific primers. In order not to bias our screen towards a specific developmental stage, genomic DNA rather than cDNA was used as the template for amplification. Degenerate oligonucleotide primers complimentary to evolutionarily conserved regions of the PTK catalytic domain were designed after Wilks (PNAS USA 86:1603-7, 1989), with the modification that inosine was introduced at locations where all four bases were possible (fig.1). ........................................................ fig.1 Degenerate oligonucleotide PCR primers. (i=inosine) . HRDLAARN (Hanks motif VIb) 5' CCGAATTCCA(C/T)(C/A)GIGA(C/T)(C/T)TIGCIGCI(C/A)GIAA 3' . DVWS(F/Y)GV (partial Hanks motif IX) 5' CCGAATTCIACICC(A/G)(A/T)AI(G/C)(A/T)CCAIAC(G/A)TC 3' .............................................................. A complex library of kinase-related sequences has been constructed by subcloning two distinct sizes of product of approximately 220bp and 300bp into pBluescript. Sequencing of 42 randomly chosen clones from the library has revealed 14 distinct PTK genes, of which 10 have not been previously described. CeHD-9,-13,-17, and-15, were sequenced as part of the genome project and can be found on contigs b0523, M142, c01G6, and M79 respectively. Predicted introns occur in 8 of the 14 genes, varying in length fom 50 to 80 bp, but only interrupting the coding sequence at four discrete locations. Shared intron position was highly predictive of sequence similarity in the case of CeHD-5 and -7 and in the case of CeHD-9,-13, and -22. The library is currently being exhaustively screened, and the genes are being mapped to the Cambridge YAC polytene grids (many thanks to Alan Coulson). These data will be presented at a later date. ................................................................ Predicted translation of kinase gene fragments. ................................................................. clone number 9 CLYGDG-K--VKISDFGLTR----RGTIYQLH---PETKSPIRWLAVETIRTMIRLLS-KT 22 CLYTDG-K--VKISDFGLTR----NGTVYEIK---PNTKSPIRWLAIETIKTMICS-E-NT 13 CLYGDG-K--VKIXDFGLTR----DGTIYQIK---PNTKAPIRWLAVEADEIHVYS-Q-KS 3 CLITKELN--VKISDFGLSV----NESETKMK---SLKKAPIRWLSPETFSKGLFN-E-KT 8 CLIS--FDGIVKIADFGLSKTLEKDQKAFKEALKEA----PPAWLAPECIQRE-SEFSTKT 17 CLVG-DTRT-IKIADFGLMRT-SYGSDYYKML---HRSWMPVRWMSKEAIEQGRFS-E-AA 20 ILLARDERT-VKICDFGLMRALKENEQMYTMA---PQKKVPFTWCPPEALRHRKFSSH-AS 28 ILVCSP--QCVKLADFGLSRALD-----YDAVYTARSGKLPIKWLAPE-VNYR-Q-FSMAS 41 ILVFSK--DKVKISDFGLSRALGVGKDYYKTNFNV-NLKLPIAWCAPECINYL-R-FTNAS 4 ILINNSLSV--KIADFGLARIL-MKENEYEA--RTGGARFPIKWTGPEAANYN-R-FTTKS 39 VLVGDKISGVPKVADFGLARKL-MEEDIYEA--RTG-AKFPIKWTAPEAATCG-N-FTVKS 15 CLVSEH--NIVKIADFGLAR-F-MKEDTYTA--HAG-AKFPIKWTAPEA--FN-T-FSSKS 5 VFVKR--NKMIRIGDFGLARHHS-KKSYYRMQCNPDTP-LPIFWLAPECFNES--KF-TES 7 VLIKR--NGVIRIADFGLARRHE-NKDYYRTR-SVGTA-IPLNWLAPECFEGSINKFDSKS