The relationship between intraflagellar transport and upstream protein trafficking pathways and macrocyclic lactone resistance in Caenorhabditis elegans.

Parasitic nematodes are globally important and place a heavy disease burden on infected humans, crops, and livestock, while commonly administered anthelmintics used for treatment are being rendered ineffective by increasing levels of resistance. It has recently been shown in the model nematode Caenorhabditis elegans that the sensory cilia of the amphid neurons play an important role in resistance toward macrocyclic lactones such as ivermectin (an avermectin) and moxidectin (a milbemycin) either through reduced uptake or intertissue signaling pathways. This study interrogated the extent to which ciliary defects relate to macrocyclic lactone resistance and dye-filling defects using a combination of forward genetics and targeted resistance screening approaches and confirmed the importance of intraflagellar transport in this process. This approach also identified the protein trafficking pathways used by the downstream effectors and the components of the ciliary basal body that are required for effector entry into these nonmotile structures. In total, 24 novel C. elegans anthelmintic survival-associated genes were identified in this study. When combined with previously known resistance genes, there are now 46 resistance-associated genes that are directly involved in amphid, cilia, and intraflagellar transport function.

The long-distance relationship between Dirofilaria and the UK: case report and literature review.

Over the last two decades, vector-borne pathogens (VBPs) have changed their distribution across the globe as a consequence of a variety of environmental, socioeconomic and geopolitical factors. Dirofilaria immitis and Dirofilaria repens are perfect exemplars of European VBPs of One Health concern that have undergone profound changes in their distribution, with new hotspots of infection appearing in previously non-endemic countries. Some areas, such as the United Kingdom, are still considered non-endemic. However, a combination of climate change and the potential spread of invasive mosquito species may change this scenario, exposing the country to the risk of outbreaks of filarial infections. Only a limited number of non-autochthonous cases have been recorded in the United Kingdom to date. These infections remain a diagnostic challenge for clinicians unfamiliar with these "exotic" parasites, which in turn complicates the approach to treatment and management. Therefore, this review aims to (i) describe the first case of D. repens infection in a dog currently resident in Scotland, (ii) summarise the available literature on Dirofilaria spp. infections in both humans and animals in the United Kingdom and (iii) assess the suitability of the United Kingdom for the establishment of these new VBPs.

Dietary-derived vitamin B12 protects Caenorhabditis elegans from thiol-reducing agents.

BACKGROUND: One-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. RESULTS: We describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, ß-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels. CONCLUSIONS: This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.

The golden death bacillus Chryseobacterium nematophagum is a novel matrix digesting pathogen of nematodes.

BACKGROUND: Nematodes represent important pathogens of humans and farmed animals and cause significant health and economic impacts. The control of nematodes is primarily carried out by applying a limited number of anthelmintic compounds, for which there is now widespread resistance being reported. There is a current unmet need to develop novel control measures including the identification and characterisation of natural pathogens of nematodes. RESULTS: Nematode killing bacilli were isolated from a rotten fruit in association with wild free-living nematodes. These bacteria belong to the Chryseobacterium genus (golden bacteria) and represent a new species named Chryseobacterium nematophagum. These bacilli are oxidase-positive, flexirubin-pigmented, gram-negative rods that exhibit gelatinase activity. Caenorhabditis elegans are attracted to and eat these bacteria. Within 3 h of ingestion, however, the bacilli have degraded the anterior pharyngeal chitinous lining and entered the body cavity, ultimately killing the host. Within 24 h, the internal contents of the worms are digested followed by the final digestion of the remaining cuticle over a 2-3-day period. These bacteria will also infect and kill bacterivorous free-living (L1-L3) stages of all tested parasitic nematodes including the important veterinary Trichostrongylids such as Haemonchus contortus and Ostertagia ostertagi. The bacteria exhibit potent collagen-digesting properties, and genome sequencing has identified novel metalloprotease, collagenase and chitinase enzymes representing potential virulence factors. CONCLUSIONS: Chryseobacterium nematophagum is a newly discovered pathogen of nematodes that rapidly kills environmental stages of a wide range of key nematode parasites. These bacilli exhibit a unique invasion process, entering the body via the anterior pharynx through the specific degradation of extracellular matrices. This bacterial pathogen represents a prospective biological control agent for important nematode parasites.

The sensory amphidial structures of Caenorhabditis elegans are involved in macrocyclic lactone uptake and anthelmintic resistance.

Parasitic nematodes represent formidable pathogens of humans, livestock and crop plants. Control of these parasites is almost exclusively dependent on a small group of anthelmintic drugs, the most important of which belong to the macrocyclic lactone class. The extensive use of these drugs to control the ubiquitous trichostrongylid parasites of grazing livestock has resulted in the emergence of both single and multi-drug resistance. The expectation is that this resistance will eventually occur in the human parasites such as the common and debilitating soil transmitted nematodes and vector-borne filarial nematodes. While the modes of action of anthelmintics such as ivermectin, have been elucidated, notably in the model nematode Caenorhabditis elegans, the molecular nature of this resistance remains to be fully determined. Here we show that the anterior amphids play a key role in ivermectin uptake and mutations in these sensory structures result in ivermectin resistance in C. elegans. Random genetic mutant screens, detailed analysis of existing amphid mutants and lipophilic dye uptake indicate that the non-motile ciliated amphid neurons are a major route of ivermectin ingress; the majority of the mutants characterised in this study are predicted to be involved in intraflagellar transport. In addition to a role in ivermectin resistance, a subset of the amphid mutants are resistant to the non-related benzimidazole class of anthelmintics, raising the potential link to a multi-drug resistance mechanism. The amphid structures are present in all nematodes and are clearly defined in a drug-sensitive strain of Haemonchus contortus. It is predicted that amphidial drug uptake and intraflagellar transport may prove to be significant in the development of single and multi-drug resistance in the nematode pathogens of veterinary and human importance.

DEMONSTRATION OF THE ANTHELMINTIC POTENCY OF MARIMASTAT IN THE HELIGMOSOMOIDES POLYGYRUS RODENT MODEL.

In the course of a structure based drug discovery program the known anticancer candidate marimastat was uncovered as a potent inhibitor of an enzyme in nematode cuticle biogenesis. It was shown to kill Caenorhabditis elegans, and the sheep parasites Haemonchus contortus and Teladorsagia circumcinta via an entirely novel nematode-specific pathway, specifically by inhibiting cuticle-remodelling enzymes that the parasites require for the developmentally essential moulting process. This discovery prompted an investigation of the compound's effect on Heligmosomoides polygyrus parasites in a mouse model of helminth infection. Mice were administered the drug via oral gavage daily from day of infection for a period of 2 wk. A second group received the drug via intra-peritoneal implantation of an osmotic minipump for 4 wk. Control groups were administered identical volumes of water by oral gavage in both cases. Counts of H. polygyrus faecal egg and larval load showed that marimastat effected a consistent and significant reduction in egg laying, and a consistent but minor reduction in adult worm load when administered every day, starting on the first day of infection. However, the drug failed to have any significant effect on egg counts or worm burdens when administered to mice with established infections. Therefore, marimastat does not appear to show promise as an anthelmintic in gastrointestinal nematode infections, although other metalloproteases such as batimastat may prove more effective.

Identification and activity of inhibitors of the essential nematode-specific metalloprotease DPY-31.

Infection by parasitic nematodes is widespread in the developing world causing extensive morbidity and mortality. Furthermore, infection of animals is a global problem, with a substantial impact on food production. Here we identify small molecule inhibitors of a nematode-specific metalloprotease, DPY-31, using both known metalloprotease inhibitors and virtual screening. This strategy successfully identified several µM inhibitors of DPY-31 from both the human filarial nematode Brugia malayi, and the parasitic gastrointestinal nematode of sheep Teladorsagia circumcincta. Further studies using both free living and parasitic nematodes show that these inhibitors elicit the severe body morphology defect 'Dumpy' (Dpy; shorter and fatter), a predominantly non-viable phenotype consistent with mutants lacking the DPY-31 gene. Taken together, these results represent a start point in developing DPY-31 inhibition as a totally novel mechanism for treating infection by parasitic nematodes in humans and animals.

A highly conserved, inhibitable astacin metalloprotease from Teladorsagia circumcincta is required for cuticle formation and nematode development.

Parasitic nematodes cause chronic, debilitating infections in both livestock and humans worldwide, and many have developed multiple resistance to the currently available anthelmintics. The protective collagenous cuticle of these parasites is required for nematode survival and its synthesis has been studied extensively in the free-living nematode, Caenorhabditis elegans. The collagen synthesis pathway is a complex, multi-step process involving numerous key enzymes, including the astacin metalloproteases. Nematode astacinsare crucial for C. elegans development, having specific roles in hatching, moulting and cuticle synthesis. NAS-35 (also called DPY-31) is a homologue of a vertebrate procollagen C-proteinase and performs a central role in cuticle formation of C. elegans as its mutation causes temperature-sensitive lethality and cuticle defects. The characterisation of DPY-31 from the ovine gastrointestinal nematode Teladorsagia circumcincta and its ability to rescue the C. elegans mutant is described. Compounds with a hydroxamate functional group have previously been shown to be potent inhibitors of procollagen C-proteinases and were therefore examined for inhibitory activity against the T. circumcincta enzyme. Phenotypic screening against T. circumcincta, Haemonchus contortus and C. elegans larval stages identified compounds that caused body morphology phenotypes consistent with the inhibition of proteases involved in cuticle collagen synthesis. These compounds correspondingly inhibited the activity of recombinant T. circumcincta DPY-31, supporting the hypothesis that this enzyme may represent a potentially novel anthelmintic drug target.

Enzymology of the nematode cuticle: A potential drug target?

All nematodes possess an external structure known as the cuticle, which is crucial for their development and survival. This structure is composed primarily of collagen, which is secreted from the underlying hypodermal cells. Extensive studies using the free-living nematode Caenorhabditis elegans demonstrate that formation of the cuticle requires the activity of an extensive range of enzymes. Enzymes are required both pre-secretion, for synthesis of component proteins such as collagen, and post-secretion, for removal of the previous developmental stage cuticle, in a process known as moulting or exsheathment. The excretion/secretion products of numerous parasitic nematodes contain metallo-, serine and cysteine proteases, and these proteases are conserved across the nematode phylum and many are involved in the moulting/exsheathment process. This review highlights the enzymes required for cuticle formation, with a focus on the post-secretion moulting events. Where orthologues of the C. elegans enzymes have been identified in parasitic nematodes these may represent novel candidate targets for future drug/vaccine development.

Prolyl 4-hydroxlase activity is essential for development and cuticle formation in the human infective parasitic nematode Brugia malayi.

Collagen prolyl 4-hydroxylases (C-P4H) are required for formation of extracellular matrices in higher eukaryotes. These enzymes convert proline residues within the repeat regions of collagen polypeptides to 4-hydroxyproline, a modification essential for the stability of the final triple helix. C-P4H are most often oligomeric complexes, with enzymatic activity contributed by the α subunits, and the ß subunits formed by protein disulfide isomerase (PDI). Here, we characterize this enzyme class in the important human parasitic nematode Brugia malayi. All potential C-P4H subunits were identified by detailed bioinformatic analysis of sequence databases, function was investigated both by RNAi in the parasite and heterologous expression in Caenorhabditis elegans, whereas biochemical activity and complex formation were examined via co-expression in insect cells. Simultaneous RNAi of two B. malayi C-P4H α subunit-like genes resulted in a striking, highly penetrant body morphology phenotype in parasite larvae. This was replicated by single RNAi of a B. malayi C-P4H ß subunit-like PDI. Surprisingly, however, the B. malayi proteins were not capable of rescuing a C. elegans α subunit mutant, whereas the human enzymes could. In contrast, the B. malayi PDI did functionally complement the lethal phenotype of a C. elegans ß subunit mutant. Comparison of recombinant and parasite derived material indicates that enzymatic activity may be dependent on a non-reducible covalent link, present only in the parasite. We therefore demonstrate that C-P4H activity is essential for development of B. malayi and uncover a novel parasite-specific feature of these collagen biosynthetic enzymes that may be exploited in future parasite control.

Design and synthesis of conformationally constrained cyclophilin inhibitors showing a cyclosporin-A phenotype in C. elegans.

Cyclophilin A (CypA) is a member of the immunophilin family of proteins and receptor for the immunosuppressant drug cyclosporin A (CsA). Here we describe the design and synthesis of a new class of small-molecule inhibitors for CypA that are based upon a dimedone template. Electrospray mass spectrometry is utilised as an initial screen to quantify the protein affinity of the ligands. Active inhibitors and fluorescently labelled derivatives are then used as chemical probes for investigating the biological role of cyclophilins in the nematode Caenorhabditis elegans.

The astacin metalloprotease moulting enzyme NAS-36 is required for normal cuticle ecdysis in free-living and parasitic nematodes.

Nematodes represent one of the most abundant and species-rich groups of animals on the planet, with parasitic species causing chronic, debilitating infections in both livestock and humans worldwide. The prevalence and success of the nematodes is a direct consequence of the exceptionally protective properties of their cuticle. The synthesis of this cuticle is a complex multi-step process, which is repeated 4 times from hatchling to adult and has been investigated in detail in the free-living nematode, Caenorhabditis elegans. This process is known as moulting and involves numerous enzymes in the synthesis and degradation of the collagenous matrix. The nas-36 and nas-37 genes in C. elegans encode functionally conserved enzymes of the astacin metalloprotease family which, when mutated, result in a phenotype associated with the late-stage moulting defects, namely the inability to remove the preceding cuticle. Extensive genome searches in the gastrointestinal nematode of sheep, Haemonchus contortus, and in the filarial nematode of humans, Brugia malayi, identified NAS-36 but not NAS-37 homologues. Significantly, the nas-36 gene from B. malayi could successfully complement the moult defects associated with C. elegans nas-36, nas-37 and nas-36/nas-37 double mutants, suggesting a conserved function for NAS-36 between these diverse nematode species. This conservation between species was further indicated when the recombinant enzymes demonstrated a similar range of inhibitable metalloprotease activities.

Selective chemical intervention in the proteome of Caenorhabditis elegans.

We present the first study of protein regulation by ligands in Caenorhabditis elegans. The ligands were peptidyl-prolyl isomerase inhibitors of cyclophilins. Up-regulation is observed for several heat shock proteins and one ligand in particular caused a greater than 2-fold enhancement of cyclophilin CYN-5. Additionally, several metabolic enzymes display elevated levels. This approach, using label-free relative quantification, provides an extremely attractive way of measuring the effect of ligands on an entire proteome, with minimal sample pretreatment, which could be applicable to large-scale studies. In this initial study, which compares the effect of three ligands, 54 unique proteins have been identified that are up- (51) or down- (3) regulated in the presence of a given ligand. A total of 431 C. elegans proteins were identified. Our methodology provides an intriguing new direction for in vivo screening of the effects of novel and untested ligands at the whole organism level.

Collagen processing and cuticle formation is catalysed by the astacin metalloprotease DPY-31 in free-living and parasitic nematodes.

The exoskeleton or cuticle performs many key roles in the development and survival of all nematodes. This structure is predominantly collagenous in nature and requires numerous enzymes to properly fold, modify, process and cross-link these essential structural proteins. The cuticle structure and its collagen components are conserved throughout the nematode phylum but differ from the collagenous matrices found in vertebrates. This structure, its formation and the enzymology of nematode cuticle collagen biogenesis have been elucidated in the free-living nematode Caenorhabditis elegans. The dpy-31 gene in C. elegans encodes a procollagen C-terminal processing enzyme of the astacin metalloprotease or bone morphogenetic protein class that, when mutated, results in a temperature-sensitive lethal phenotype associated with cuticle defects. In this study, orthologues of this essential gene have been identified in the phylogenetically diverse parasitic nematodes Haemonchus contortus and Brugia malayi. The DPY-31 protein is expressed in the gut and secretory system of C. elegans, a location also confirmed when a B. malayi transcriptional dpy-31 promoter-reporter gene fusion was expressed in C. elegans. Functional conservation between the nematode enzymes was supported by the fact that heterologous expression of the H. contortus dpy-31 orthologue in a C. elegans dpy-31 mutant resulted in the full rescue of the mutant body form. This interspecies conservation was further established when the recombinant nematode enzymes were found to have a similar range of inhibitable protease activities. In addition, the recombinant DPY-31 enzymes from both H. contortus and B. malayi were shown to efficiently process the C. elegans cuticle collagen SQT-3 at the correct C-terminal procollagen processing site.

The kunitz domain protein BLI-5 plays a functionally conserved role in cuticle formation in a diverse range of nematodes.

The cuticle of parasitic nematodes performs many critical functions and is essential for proper development and for protection from the host immune response. The biosynthesis, assembly, modification and turnover of this exoskeleton have been most extensively studied in the free-living nematode, Caenorhabditis elegans, where it represents a complex multi-step process involving a whole suite of enzymes. The biosynthesis of the cuticle has an additional level of complexity, as many of the enzymes also require additional proteins to aid their activation and selective inhibition. Blister-5 (BLI-5) represents a protein with a kunitz-type serine protease interacting domain and is involved in cuticle collagen biosynthesis in C. elegans, through its interaction with subtilisin-like processing enzymes (such as BLI-4). Mutation of the bli-5 gene causes blistering of the collagenous adult cuticle. Homologues of BLI-5 have been identified in several parasitic species that span different nematode clades. In this study, we molecularly and biochemically characterize BLI-5 homologues from the clade V nematodes C. elegans and Haemonchus contortus and from the clade III filarial nematode Brugia malayi. The nematode BLI-5 orthologues possess a shared domain structure and perform similar in vitro and in vivo functions, performing important proteolytic enzyme functions. The results demonstrate that the bli-5 genes from these diverse parasitic nematodes are able to complement a C. elegansbli-5 mutant and thereby support the use of the C. elegans model system to examine gene function in the experimentally less-amenable parasitic species.

Combined extracellular matrix cross-linking activity of the peroxidase MLT-7 and the dual oxidase BLI-3 is critical for post-embryonic viability in Caenorhabditis elegans.

The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as molting. A targeted RNA interference screen using a cuticle collagen marker has been employed to identify components of the cuticle biosynthetic pathway. We have characterized an essential peroxidase, MoLT-7 (MLT-7), that is responsible for proper cuticle molting and re-synthesis. MLT-7 is an active, inhibitable peroxidase that is expressed in the cuticle-synthesizing hypodermis coincident with each larval molt. mlt-7 mutants show a range of body morphology defects, most notably molt, dumpy, and early larval stage arrest phenotypes that can all be complemented with a wild type copy of mlt-7. The cuticles of these mutants lacks di-tyrosine cross-links, becomes permeable to dye and accessible to tyrosine iodination, and have aberrant collagen protein expression patterns. Overexpression of MLT-7 causes mutant phenotypes further supporting its proposed enzymatic role. In combination with BLI-3, an H2O2-generating NADPH dual oxidase, MLT-7 is essential for post-embryonic development. Disruption of mlt-7, and particularly bli-3, via RNA interference also causes dramatic changes to the in vivo cross-linking patterns of the cuticle collagens DPY-13 and COL-12. This points toward a functionally cooperative relationship for these two hypodermally expressed proteins that is essential for collagen cross-linking and proper extracellular matrix formation.

Four abundant novel transcript genes from Toxocara canis with unrelated coding sequences share untranslated region tracts implicated in the control of gene expression.

The Toxocara canis "abundant novel transcripts" (ant) are four highly expressed products, constituting >18% of ESTs from the infective stage of this widely prevalent nematode parasite. Using 5' RACE, we determined full-length sequences for each ant gene, between 1.8 and 2.8kb. The four genes (termed ant-3, -5, -30 and -34), share no coding sequence similarity, although their 3'UTRs (untranslated regions) are homologous. Predicted ANT-5 and ANT-30 proteins show distant similarity to RNA regulatory proteins, RNA-dependent RNA polymerase and DEAH-box helicase, respectively. Surprisingly, ant-3 appears to be bi-cistronic, encoding two ORFs (ANT-3.1 and -3.2), each with a predicted N-terminal signal sequence. Antibodies raised to recombinant proteins did not react with native parasite products, indicating that protein expression did not accord with transcript abundance. However, antibody reactivity to two gene products (ANT-3.1 and ANT-34) was present in patient sera, suggesting that these proteins are synthesized later in infection. To test whether 3'UTRs may regulate expression, the ant-34 3'UTR sequence was inserted adjacent to enhanced green fluorescent protein (EGFP) for transformation of Caenorhabditis elegans. The ant-34 3'UTR greatly reduced EGFP expression, inhibiting both transcription and translation. We identified a tract in this UTR with significant sequence complementarity to the C. elegans micro-RNA lin-4. While infective stage parasites stockpile high levels of the ant transcripts, we suggest that translation is repressed, possibly by a mechanism involving 3' UTR motifs shared by the four genes.

Characterization of a novel Caenorhabditis elegans prolyl 4-hydroxylase with a unique substrate specificity and restricted expression in the pharynx and excretory duct.

Collagen prolyl 4-hydroxylases (C-P4Hs) have a critical role in collagen synthesis, since 4-hydroxyproline residues are necessary for folding of the triple-helical molecules. Vertebrate C-P4Hs are alpha(2)beta(2) tetramers in which the beta subunit is identical to protein-disulfide isomerase (PDI). Three isoforms of the catalytic alpha subunit, PHY-1, PHY-2, and PHY-3, have been characterized from Caenorhabditis elegans, PHY-1 and PHY-2 being responsible for the hydroxylation of cuticle collagens, whereas PHY-3 is predicted to be involved in collagen synthesis in early embryos. We have characterized transcripts of two additional C. elegans alpha subunit-like genes, Y43F8B.4 and C14E2.4. Three transcripts were generated from Y43F8B.4, and a polypeptide encoded by one of them, named PHY-4.1, assembled into active (PHY-4.1)(2)/(PDI-2)(2) tetramers and PHY-4.1/PDI-2 dimers when coexpressed with C. elegans PDI-2 in insect cells. The C14E2.4 transcript was found to have a frameshift leading to the absence of codons for two residues critical for P4H catalytic activity. Thus, C. elegans has altogether four functional C-P4H alpha subunits, PHY-1, PHY-2, PHY-3, and PHY-4.1. The tetramers and dimers containing recombinant PHY-4.1 had a distinct substrate specificity from the other C-P4Hs in that they hydroxylated poly(l-proline) and certain other proline-rich peptides, including ones that are expressed in the pharynx, in addition to collagen-like peptides. These data and the observed restricted expression of the phy-4.1 transcript and PHY-4.1 polypeptide in the pharyngeal gland cells and the excretory duct suggest that in addition to collagens, PHY-4.1 may hydroxylate additional proline-rich proteins in vivo.

Cloning, purification and characterization of the Caenorhabditis elegans small glutamine-rich tetratricopeptide repeat-containing protein.

We have cloned and expressed the putative Caenorhabditis elegans orthologue for small glutamine-rich tetratricopeptide repeat-containing protein, now assigned the gene name sgt-1 in the C. elegans genome database. Characterization of the purified protein by cross-linking, mass spectrometry and gel filtration experiments provides unambiguous evidence that SGT-1 forms homo-dimers in solution. The hydrodynamic dimensions of SGT-1 dimers in relation to their molecular weight suggest a protein with a low level of compactness and an extended conformation. Human SGT has been shown to interact with and regulate the activity of heat shock proteins Hsp70 and Hsp90 via a TPR domain mediated interaction. The SGT TPR domain (SGT-1-TPR, residues 100-226) was cloned, purified and shown by ITC and CD analysis to interact with the C-terminal peptides of Hsp70 and Hsp90 with comparable affinities although there is no evidence of a recently proposed coupled binding-folding mechanism for TPR domains.

The cuticle.

The nematode cuticle is an extremely flexible and resilient exoskeleton that permits locomotion via attachment to muscle, confers environmental protection and allows growth by molting. It is synthesised five times, once in the embryo and subsequently at the end of each larval stage prior to molting. It is a highly structured extra-cellular matrix (ECM), composed predominantly of cross-linked collagens, additional insoluble proteins termed cuticlins, associated glycoproteins and lipids. The cuticle collagens are encoded by a large gene family that are subject to strict patterns of temporal regulation. Cuticle collagen biosynthesis involves numerous co- and post-translational modification, processing, secretion and cross-linking steps that in turn are catalysed by specific enzymes and chaperones. Mutations in individual collagen genes and their biosynthetic pathway components can result in a range of defects from abnormal morphology (dumpy and blister) to embryonic and larval death, confirming an essential role for this structure and highlighting its potential as an ECM experimental model system.