Chitwood DJ et al. (1987) Comparative Biochemistry and Physiology "Sterol metabolism in the nematodes Panagrellus redivivus, Turbatrix aceti and C. elegans."

Chitwood DJ, Salt TA, Lusby WR

[Comparative Biochemistry and Physiology, 1987]

1. Panagrellus redivivus, Turbatrix aceti and Caenorhabditis elegans were sterilely propagated in semidefined media containing sitosterol or cholesterol, and sterols were isolated and identifed by capillary gas-liquid chromatography-mass spectometry. 2. Each species was capable of removal of the C-24 ethyl substituent of sitosterol and production of 4a-methylsterols. 3. Other modifications of the sterol nucleus varied among the species, as only T. aceti and C. elegans introduced *7- and *8(14)-bonds significantly. 4. P. redivivus and, to a much lesser extent, T. aceti reduced *5-bonds to produce substantial quantities of cholesterol and 4a-methylcholestanol.

Wang C et al. (2018) Chem Sci "Reductive cleavage of C[double bond, length as m-dash]C bonds as a new strategy for turn-on dual fluorescence in effective sensing of H2S."

Zhang X, Ding Z, Li G, Zhang H, Yuan D, Wang C, Tan J, Wang W, Cheng X

[Chem Sci, 2018]

Reductive cleavage of alkenes is rarely reported in synthetic chemistry. Here we report a unique H₂S-mediated reductive cleavage of C[double bond, length as m-dash]C bonds under mild conditions, which is a successful new strategy for the design of probes for effective sensing of H₂S with turn-on dual-color fluorescence. A short series of phenothiazine ethylidene malononitrile derivatives were shown to react with H₂S, <i>via</i> reductive cleavage of C[double bond, length as m-dash]C bonds with intramolecular cyclization reactions to form thiophene rings. Enlightened by this new reaction mechanism, four effective probes with turn-off to turn-on fluorescence switches were successfully applied for sensing H₂S, an important gaseous signalling molecule in living systems, among which PTZ-P4 exhibited two fluorescent colors after reductive cleavage. The dual-color probe was applied for imaging endogenous H₂S and showed distinct differences in brightness in living <i>C. elegans</i> for wild type N2, <i>glp-1</i> (<i>e2144</i>) mutants (higher levels of endogenous H₂S), and <i>cth-1</i> (<i>ok3319</i>) mutants (lower levels of endogenous H₂S). The discovery of H₂S-mediated reductive cleavage of C[double bond, length as m-dash]C bonds is expected to be valuable for chemical synthesis, theoretical studies, and the design of new fluorescent H₂S probes.

Shi L et al. (2018) Nat Commun "Optical imaging of metabolic dynamics in animals."

Zhang L, Chen Z, Silveira ES, Targoff K, Shi L, Zheng C, Min W, de Sena-Tomas C, Shen Y, Wei M, Liu C

[Nat Commun, 2018]

Direct visualization of metabolic dynamics in living animals with high spatial and temporal resolution is essential to understanding many biological processes. Here we introduce a platform that combines deuterium oxide (D₂O) probing with stimulated Raman scattering (DO-SRS) microscopy to image in situ metabolic activities. Enzymatic incorporation of D₂O-derived deuterium into macromolecules generates carbon-deuterium (C-D) bonds, which track biosynthesis in tissues and can be imaged by SRS in situ. Within the broad vibrational spectra of C-D bonds, we discover lipid-, protein-, and DNA-specific Raman shifts and develop spectral unmixing methods to obtain C-D signals with macromolecular selectivity. DO-SRS microscopy enables us to probe de novo lipogenesis in animals, image protein biosynthesis without tissue bias, and simultaneously visualize lipid and protein metabolism and reveal their different dynamics. DO-SRS microscopy, being noninvasive, universally applicable, and cost-effective, can be adapted to a broad range of biological systems to study development, tissue homeostasis, aging, and tumor heterogeneity.

Cox GN et al. (1981) J Cell Biol "Cuticle of Caenorhabditis elegans: its isolation and partial characterization."

Edgar RS, Kusch M, Cox GN

[J Cell Biol, 1981]

The adult cuticle of the soil nematode, Caenorhabditis elegans, is a proteinaceous extracellular structure elaborated by the underlying layer of hypodermal cells during the final molt in the animal's life cycle. The cuticle is composed of an outer cortical layer connected by regularly arranged struts to an inner basal layer. The cuticle can be isolated largely intact and free of all cellular material by sonication and treatment with 1% sodium dodecyl sulfate (SDS). Purified cuticles exhibit a negative material in the basal cuticle layer. The cuticle layers differ in their solubility in sulfhydryl reducing agents, susceptibility to various proteolytic enzymes and amino acid composition. The struts, basal layer, and internal cortical layer are composed of collagen proteins that are extensively cross-linked by disulfide bonds. The external cortical layer appears to contain primarily noncollagen proteins that are extensively cross- linked by nonreducible covalent bonds. The collagen proteins extracted from the cuticle with a reducing agent can be separated by SDS-polyacrylamide gel electrophoresis into eight major species differing in apparent molecular weight.

Wilson WR et al. (1994) Mol Biochem Parasitol "The Onchocerca volvulus homologue of the multifunctional polypeptide protein disulfide isomerase."

Shepley KJ, Tuan RS, Greene BM, Wilson WR, Unnasch TR, Freedman DO, Awadzi K

[Mol Biochem Parasitol, 1994]

Protein disulfide isomerase (PDI) functions to catalyze the formation of correct disulfide bonds in nascent proteins, and also acts as one of the subunits of prolyl-4 hydroxylase, the enzyme responsible for the oxidative maturation of procollagen. Since the cuticle of parasitic nematodes consists primarily of a network of collagen molecules which are connected through intermolecular disulfide bonds, PDI might be expected to be involved in the process of cuticle biosynthesis. The isolation and characterization of a cDNA encoding the PDI homologue of Onchocerca volvulus is described. This cDNA contains a single, long open reading frame that encodes sequence motifs identical to the two known active sites of PDI for isomerase activity. The O. volvulus PDI appears to be encoded by a single copy gene. Both in situ hybridization and immunolocalization data suggest that PDI is both spatially and temporally regulated in O. volvulus. The pattern of spatial and temporal regulation is consistent with the involvement of PDI in the biosynthesis of the parasite cuticle. The parasite protein appears to be an antigen recognized by a minority of individuals exposed to O. volvulus.

Conraths FJ et al. (1997) Exp Parasitol "Expression of the microfilarial sheath protein 2 (shp2) of the filarial parasites Litomosoides sigmodontis and Brugia malayi."

Hobom G, Zahner H, Conraths FJ, Hirzmann J

[Exp Parasitol, 1997]

The microfilarial sheaths of the filarial parasites Brugia malayi, Brugia pahangi, and Litomosoides sigmodontis consist of several parasite proteins, probably ranging between 7 and 10. The gene encoding sheath protein 2 (shp2), which is the object of this study, is transcribed in embryos and in the uterine epithelium; at least in B. malayi, it is translated in both tissues. Apparently, shp2 is synthesized as a monomer, exported by the respective cells, and integrated into the microfilarial sheath. In the sheath, it exists as a highly polymerized molecule cross-linked by cysteine formation and other covalent bonds, presumably epsilon-(gamma-glutamyl)-lysine links.

McNally FJ et al. (1993) Cell "Identification of katanin, an ATPase that severs and disassembles stable microtubules."

McNally FJ, Vale RD

[Cell, 1993]

Eukaryotic cells rapidly reorganize their microtubule cytoskeleton during the cell cycle, differentiation, and cell migration. In this study, we have purified a heterodimeric protein, katanin, that severs and disassembles microtubules to tubulin dimers. The disassembled tubulin can repolymerize, indicating that it is not irreversibly modified or denatured in the reaction. Katanin is a microtubule-stimulated ATPase and requires ATP hydrolysis to sever microtubules. Katanin represents a novel type of enzyme that utilizes energy from nucleotide hydrolysis to break tubulin-tubulin bonds within a microtubule polymer, a process that may aid in disassembling complex microtubule arrays within cells.

Yang J et al. (1999) J Biol Chem "Proteolytic processing of Caenorhabditis elegans SQT-1 cuticle collagen is inhibited in right roller mutants whereas cross-linking is inhibited in left roller mutants."

Yang J, Kramer JM

[J Biol Chem, 1999]

The sqt-1 gene encodes a C. elegans cuticle collagen that when defective can cause dramatic alterations of organismal morphology. Specific antisera were used to examine the assembly of wild-type and mutant SQT-1 in the cuticle. Wild-type SQT-1 chains associate into dimer, tetramer, and higher oligomers that are cross-linked by non-reducible, presumably tyrosine-derived, covalent bonds. The SQT-1 pattern differs from the bulk of cuticle collagens which are found in trimer and larger forms. sqt-1 mutations that cause left-handed helical twisting of animals remove a conserved carboxyl-domain cysteine and inhibit formation of these non-reducible bonds. SQT-1 monomers accumulate and novel trimer-sized products form. A conserved tyrosine immediately adjacent to the affected cysteine suggests that disulfide bond formation is required for this tyrosine to form a cross-link. sqt-1 mutations that cause right-handed helical twisting affect conserved arginines in a predicted cleavage site for a subtilisin-like protease. These mutant SQT-1 molecules retain residues on the amino side of the predicted cleavage site and are larger than wild-type by the amount expected if cleavage failed to occur. The conservation of this site in all nematode cuticle collagens indicates that they are all synthesized as procollagens that are processed by subtilisin-like proteases.

Gogonea CB et al. (1999) J Mol Graph Model "Computational prediction of the three-dimensional structures for the Caenorhabditis elegans tubulin family."

Ali YM, Gogonea V, Merz KM, Gogonea CB, Siddiqui SS

[J Mol Graph Model, 1999]

In this article we characterize, from a structural point of view, all 16 members of the tubulin gene family of Caenorhabditis elegans (9 alpha-tubulins, 6 beta-tubulins, and 1 gamma-tubulin). We obtained their tertiary structures by computationally modifying the X-ray crystal structure of the pig brain alpha/beta-tubulin dimer published by Nogales et al. [Nature (London) 1998;391:199-203]. Our computational protocol involves changing the amino acids (with MIDAS; Jarvis et al., UCSF MIDAS. University of California, San Francisco, 1986) in the 3D structure of pig brain alpha/beta-tubulin dimer followed by geometry optimization with the AMBER force field (Perlman et al., AMBER 4. University of California, San Francisco, 1990). We subsequently analyze and compare the resulting structures in terms of the differences in their secondary and tertiary structures. In addition, we compare the pattern of hydrogen bonds and hydrophobic contacts in the guanosine triphosphate (GTP)-binding site for all members of the tubulin family. Our computational results show that, except for gamma-tubulin, all members of the C. elegans tubulin family have similar secondary and 3D structures and that the change in the pattern of hydrogen bonds in the GTP-binding site may be used to assess the relative stability of different alpha/beta-tubulin dimers formed by monomers of the tubulin family.

Maurer P et al. (1997) Eur J Biochem "Structural and functional characterization of the extracellular calcium-binding protein BM-40/secreted protein, acidic, rich in cysteine/osteonectin from the nematode Caenorhabditis elegans."

Schwarzbauer JE, Mann K, Gohring W, Sasaki T, Maurer P, Timpl R

[Eur J Biochem, 1997]

Caenorhabditis elegans BM-40 (positions 19-264) and its extracellular calcium-binding domain (positions 139-264) were obtained in recombinant form from human kidney cells using an episomal expression vector. The purified proteins showed single bands of 33 kDa [BM-40-(19-264)-peptide] or 14 kDa [BM-40-(139-264)-peptide] on electrophoresis, contained internal disulfide bonds and a helices and were relatively resistant to matrix metalloproteinases. Hexosamine analysis indicated substitution by one N-linked and two O-linked oligosaccharides and recombinant BM-40 was indistinguishable in its immunological epitopes from nematode tissue-derived BM-40, suggesting that it was obtained in native form. Both recombinant C. elegans proteins showed a distinct binding activity for human collagens I and IV in solid-phase and surface-plasmon-resonance assays with an affinity (Kd = 1-2 microM), comparable to that of mammalian BM-40. However, calcium-binding studies revealed only a low-affinity site (Kd = 6.2 mM) and failed to show the characteristic conformational change upon addition of EDTA. These and a few other differences are apparently due to two extra disulfide bonds and two deletions/insertions in C. elegans BM-40 and can be partly interpreted from the X-ray structure of a large part of human BM-40. The immunological assays available and the predictions of the location of the collagen-binding epitope should facilitate a molecular and genetic approach to understand the function of BM-40 in the development of C. elegans.