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[
Biochemistry,
1987]
The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N- terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].
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[
Eur J Cell Biol,
2004]
Intestinal cells of C. elegans show an unexpectedly high complexity of cytoplasmic intermediate filament (IF) proteins. Of the 11 known IF genes six are coexpressed in the intestine, i.e. genes B2, C1, C2, D1, D2, and E1. Specific antibodies and GFP-promoter constructs show that genes B2, D1, D2, and E1 are exclusively expressed in intestinal cells. Using RNA interference (RNAi) by microinjection at 25 degreesC rather than at 20 degreesC we observe for the first time lethal phenotypes for C1 and D2. RNAi at 25 degreesC also shows that the known A1 phenotype occurs already in the late embryo after microinjection and is also observed by feeding which was not the case at 20 degreesC. Thus, RNAi at 25 degreesC may also be useful for the future analysis of other nematode genes. Finally, we show that triple RNAi at 20 degreesC is necessary for the combinations B2, D1, E1 and B2, D1, D2 to obtain a phenotype. Together with earlier results on genes A1, A2, A3, B1, and C2 RNAi phenotypes are now established for all 11 IF genes except for the A4 gene. RNAi phenotypes except for A2 (early larval lethality) and C2 (adult phenotype) relate to the late embryo. We conclude that in C. elegans cytoplasmic IFs are required for tissue integrity including late embryonic stages. This is in strong contrast to the mouse, where ablation IF genes apparently does not affect the embryo proper.
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[
Cytoskeleton (Hoboken),
2017]
The dimerisation properties of six intestine-expressed intermediate filament (IF) proteins (B2, C1, C2, D1, D2, E1) were analysed in blot overlay assay on membranes containing all of the eleven recombinant C. elegans IF proteins (A1, A2, A3, A4, B1, B2, C1, C2, D1, D2 and E1). The interactions detected in the blot assays exclusively comprise intestine-expressed IF proteins and the protein A4, which is found in the dauer larva intestine. 86% of these interactions are heterotypic, while the remaining interactions relate to C1, C2 and D2 homodimers. These multiple modes of interaction were also supported by calculations of the numbers of possible interchain ionic interactions derived from the individual rod sequences. The results predict that the six B2, C1, C2, D1, D2 and E1 IF proteins are able to form as many as eleven different heteropolymeric and three homopolymeric IFs in the C. elegans intestine. This simple model of the intestinal IF meshwork enables us to speculate that our previously reported triple RNAi worms arrested or decreased their growth because of feeding reduction due to morphological defects of the mechanically-compromised intestine. This article is protected by copyright. All rights reserved.
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[
J Mol Evol,
2019]
Our previous calculations of ionic interactions indicated that the Caenorhabditis elegans intermediate filament (IF) IFA proteins, in addition to IFA/IFB-1 heterodimers, may also form homodimers. In order to prove the significance of these calculations, we analysed the dimerization potential of the IFA chains in blot overlays. Unexpectedly, we found here that the dimerization of the IFA-1 protein was of both homotypic and heterotypic nature, and involved all proteins immobilized on the membrane (IFA-1, IFA-2, IFA-4, IFB-1, IFB-2, IFC-1, IFC-2, IFD-1, IFD-2 and IFP-1). A similar interaction profile, though less complex, was observed for two biotinylated proteins (IFA-2 and IFA-4). These and previous results indicate that the IFA proteins are able to form many different heteropolymeric and homopolymeric complexes in the C. elegans tissue, but that only those triggered by the IFA-specific IFB-1 protein result in mature IFs. Moreover, the calculations of the possible ionic interactions between the individual rod sequences as well as their various deletion variants indicated a special role in this process for the middle part of the C. elegans IF coil 1B segment that is deleted in all vertebrate cytoplasmic IFs. We hypothesized here, therefore, that the striking promiscuity of the C. elegans IFs originally involved a nuclear lamin which, due to a two-heptad-long rod deletion, prevented formation of a functional lamin/cIF dimer. This, in concert with an efficient dimerization and a strict tissue-specific co-expression, may allow expansion and maintenance of the multiple Caenorhabditis IFs. A possible implication for evolution of chordate IFs proteins is also discussed.
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Berynskyy M, Morimoto RI, Bukau B, Stengel F, Kirstein J, Szlachcic A, Arnsburg K, Stank A, Scior A, Nillegoda NB, Gao X, Guilbride DL, Aebersold R, Wade RC, Mayer MP
[
Nature,
2015]
Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.
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[
Mol Immunol,
1999]
Invertebrate cells lack the
p53 recombination checkpoint but contain mobile DNA sequences that transpose by a mechanism in part shared with excision of the V(D)J recombination signal sequences (RSS). In this work, inversion, deletion, and duplication of sequences associated with an invertebrate C. elegans Tc6 element is described. The structure of this C. elegans sequence and other dispersed Tc6 elements suggests that covalently closed 'hairpin' structures are not unique to excision of the V(D)J RSS by the RAG proteins, but rather can be generated by transposases at transposon termini leading to characteristic inversion and duplication events. Comparative analysis of recombination events at invertebrate sequences resembling the vertebrate V(D)J RSS may be useful in understanding V(D)J recombination-mediated recombination events in malignant vertebrate cells or genetic diseases such as ataxia telangectasia, in which the
p53 recombination checkpoint is defective.
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[
Phytother Res,
2008]
A bioassay-guided fractionation of Juniperus procera berries yielded antiparasitic, nematicidal and antifouling constituents, including a wide range of known abietane, pimarane and labdane diterpenes. Among these, abieta-7,13-diene (1) demonstrated in vitro antimalarial activity against Plasmodium falciparum D6 and W2 strains (IC(50) = 1.9 and 2.0 microg/mL, respectively), while totarol (6), ferruginol (7) and 7beta-hydroxyabieta-8,13-diene-11,12-dione (8) inhibited Leishmania donovani promastigotes with IC(50) values of 3.5-4.6 microg/mL. In addition, totarol demonstrated nematicidal and antifouling activities against Caenorhabditis elegans and Artemia salina at a concentration of 80 microg/mL and 1 microg/mL, respectively. The resinous exudate of J. virginiana afforded known antibacterial E-communic acid (4) and 4-epi-abietic acid (5), while the volatile oil from its trunk wood revealed large quantities of cedrol (9). Using GC/MS, the two known abietanes totarol (6) and ferruginol (7) were identified from the berries of J. procera, J. excelsa and J. phoenicea.
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[
J Mol Biol,
2003]
The in vitro polymerization and tissue-specific expression patterns of the four essential intermediate filament (IF) proteins (A1, A2, A3, B1) and the non-essential IF protein A4 were analyzed. Recombinant B1, used as a probe in blot overlay assays of the 11 Caenorhabditis elegans IF proteins, reacted strongly with proteins A1 to A4, indicating a heterotypic interaction. Obligate heteropolymeric filament assembly in vitro was confirmed by electron microscopy. Protein B1 formed long IF when mixed with an equimolar amount of A1, A2 or A3. Developmentally regulated coexpression of 131 and one or more members of the A family was found with GFP-promoter reporters. This coexpression pattern argues for a heteropolymer system in vivo. One or both splice variants of the B1 gene are always coexpressed in a tissue-specific manner with at least one member of the A family in hypodermis, pharynx, pharyngeal-intestinal valve, excretory cells, uterus, vulva and rectum. Interestingly, while the intestine normally lacks a B1/A pair, the dauer larva shows intestinal B1 and A4. These results are in line with similar postembryonic phenotypes of the hypodermis induced by RNA interference (RNAi) of genes B1, A2 and A3. Similarly, defects of the pharynx and its A1-GFP containing tonofilaments observed in the postembryonic B1 RNAi phenotype are consistent with the coexpression of B1 and A1 in the marginal cells. Thus RNAi analyses provide independent evidence for the existence of the B1/A obligate heteropolymer system in vivo. Proteins A1 and B1 have a similar and rather slow turnover rate in photobleaching experiments of the pharynx tonofilaments.
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[
J Mol Biol,
2003]
The lamins of the tunicate Ciona intestinalis and the nematode Caenorhabditis elegans show unusual sequence features when compared to the more than 35 metazoan lamin sequences currently known. We therefore analyzed the in vitro assembly of these two lamins by electron microscopy using chicken lamin B2 as a control. While lamin dimers usually appear as a rod carrying two globules at one end, these globules are absent from Ciona lamin, which lacks the central 105-residue region of the tail domain. The deletion of 14 residues or two heptads from the coiled coil rod domain of the single C. elegans lamin results in a 1.5-nm shortening of the dimer rod. Similarly, the paracrystals assembled from the C. elegans lamin exhibit a 3.1-nm reduction of the true axial repeat compared to that of chicken lamin B2 paracrystals. We speculate that the banding pattern in the C. elegans lamin paracrystals arises from a relative stagger between dimers and/or a positioning of the globular tail domain relative to the central rod that is distinct from that observed in chicken lamin B2 paracrystals. Here we show that a nuclear lamin can assemble in vitro into 10-nm intermediate filaments (IFs). C. elegans lamin in low ionic strength Tris-buffers at a pH of 7.2-7.4 provides a stable population of lamin IFs. Some implications of this filament formation are discussed.
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[
Aging Cell,
2017]
Protein aggregation is enhanced upon exposure to various stress conditions and aging, which suggests that the quality control machinery regulating protein homeostasis could exhibit varied capacities in different stages of organismal lifespan. Recently, an efficient metazoan disaggregase activity was identified invitro, which requires the Hsp70 chaperone and Hsp110 nucleotide exchange factor, together with single or cooperating J-protein co-chaperones of classes A and B. Here, we describe how the orthologous Hsp70s and J-protein of Caenorhabditis elegans work together to resolve protein aggregates both invivo and invitro to benefit organismal health. Using an RNAi knockdown approach, we show that class A and B J-proteins cooperate to form an interactive flexible network that relocalizes to protein aggregates upon heat shock and preferentially recruits constitutive Hsc70 to disaggregate heat-induced protein aggregates and polyQ aggregates that form in an age-dependent manner. Cooperation between class A and B J-proteins is also required for organismal health and promotes thermotolerance, maintenance of fecundity, and extended viability after heat stress. This disaggregase function of J-proteins and Hsc70 therefore constitutes a powerful regulatory network that is key to Hsc70-based protein quality control mechanisms in metazoa with a central role in the clearance of aggregates, stress recovery, and organismal fitness in aging.