Stepek, Gillian et al. (2015) International Worm Meeting "Nematode moulting enzymes as potential drug targets."

Houston, Doug, Walkinshaw, Malcolm, McCormack, Gillian, Page, Antony, Stepek, Gillian

[International Worm Meeting, 2015]

The nematode cuticle is a collagenous extracellular matrix that is synthesised repeatedly during development via the moulting process. The enzymology of cuticle collagen biosynthesis and more importantly the enzymology of cuticle ecdysis and moulting represent potential novel targets for parasitic nematode control. Using C. elegans we have identified and characterized key moulting enzymes and have focused on the nematode astacin (NAS) metalloproteases. As an example, mutation of the enzyme-encoding genes NAS-36 and NAS-37 reveals an essential developmental role for these enzymes in C. elegans moulting. We have identified the orthologs in the diverse parasitic nematodes Brugia malayi and Haemonchus contortus that are able to rescue the moulting phenotypes associated with nas-36/ -37.Using in silico-modelling we have screened available chemical libraries and have tested several hundred potential inhibitors using in vivo and in vitro assays in C. elegans, Brugia malayi and Haemonchus contortus.The screening process will be described and the main finding of this study will be presented.

Patterson GI et al. (1998) East Coast Worm Meeting "Signalling by daf-3 and other genes downstream of TGF-beta-related genes in dauer formation and"

Ruvkun GB, Patterson GI

[East Coast Worm Meeting, 1998]

We are interested in how environmental cues control the choice between dauer arrest and growth. We study a group of TGF-beta pathway related genes that control this process. Research performed in the labs of Don Riddle and Jim Thomas led to the cloning of genes that are homologous to TGF-beta pathways in other systems (see Fig. 1). These include daf-7 (a TGF-beta ligand), daf-1 and daf-4 (serine/threonine kinase receptors), and daf-8 and daf-14, which are homologous to the Smad family of transcription factors. Smads have been shown to be regulated by phosphorylation by the receptors in other systems. Mutations in these genes have a dauer constitutive phenotype. We have cloned the daf-3 gene, which has a dauer defective mutant phenotype that is epistatic to the dauer constitutive mutants. daf-3, like daf-8 and daf-14, is homologous to the Smad family of transcription factors. The fact that daf-3 is epistatic to daf-8 and daf-14 suggests a model in which the DAF-3 protein is negatively regulated by DAF-8 and DAF-14. In other systems, Smads proteins interact and regulate each other. We are interested in determining whether the DAF-3 protein interacts with the DAF-8 and DAF-14 proteins, and will discuss plans to study these interactions in protein extracts from C. elegans and in yeast two-hybrid experiments. In our model, the receptors function only to negatively regulate DAF-3 via DAF-8 and DAF-14. This model suggests that, unlike most Smads, DAF-3 is functional as a transcription factor without requiring phosphorylation by an upstream receptor. We will discuss experiments performed by Chang Yeol in Malcolm Whitman's lab that suggests that DAF-3 can mimic an activated Smad in Xenopus embyos. This line of experiments may allow us to verify that DAF-3 is a functional transcription factor in the absence of coupling to a receptor. In addition, we can use this system to test whether DAF-8 and DAF-14 block dauer formation by binding to DAF-3. We have isolated additional mutations that, like daf-3, suppress the dauer constitutive phenotype of mutations in the TGF-beta pathway genes. We are pursuing cloning these suppressors, which may be cofactors of DAF-3, may represent positive regulators of DAF-3 function, or may represent other genes negatively regulated by the TGF-beta pathway. Figure 1: Model for regulation of DAF-3 by a TGF-beta pathway.