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[
J Vis Exp,
2014]
Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping(1,2). Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.
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[
J Gerontol A Biol Sci Med Sci,
2021]
AMP-activated protein kinase (AMPK) is involved in life span maintenance, stress responses, and germ cell cycle arrest upon dauer entry. AMPK is currently considered a promising target for preventing age-related diseases. Rubidium is one of the trace elements in human body. As early as the 1970s, RbCl has been was reported to have neuroprotective effects. In this work, we report the anti-aging effect of RbCl in Caenorhabditis elegans. Specifically, we reveal that (1) RbCl does increase the lifespan and enhance stress resistance in C. elegans without disturbing their fecundity. (2) RbCl induces superoxide dismutase (SOD) expression, which is essential for its anti-aging and anti-stress effect. (3) AAK-2 and DAF-16 are essential to the anti-aging efficacy of RbCl, and RbCl can promote DAF-16 translocating into the nucleus, suggesting that RbCl delays aging through regulating AMPK/FOXO pathway. RbCl can be a promising agent against aging related diseases.
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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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[
J Agric Food Chem,
2020]
The aggregation of amyloid- protein (A) is deemed a vital pathological feature of Alzheimer's disease (AD). Hence, inhibiting A aggregation is noticed as a major tactic for the prevention and therapy of AD. Hydroxycinnamic acid, as a natural phenolic compound is widely present in plant foods and has several biological activities including anti-inflammation, antioxidation and neuroprotective effects. Here it was found that hydroxycinnamic acid and its structural analogues (3-hydroxycinnamic acid, 2-hydroxycinnamic acid, cinnamic acid, 3,4-dihydroxycinnamic acid, 2,4-dihydroxycinnamic acid and 3,4,5-trihydroxycinnamic acid) could inhibit A40 fibrillogenesis and reduce A40-induced cytotoxicity in a dose-dependent manner. Among these small molecules investigated, 3,4,5-trihydroxycinnamic acid is considered to be the most effective inhibitor, which reduces ThT uorescence intensity to 30.79% and increases cell viability from 49.47% to 84.78% at 200 M. And the results with C. elegans tested that these small molecules can ameliorate AD-like symptoms of worm paralysis. Moreover, molecular docking studies showed that these small molecules interact with the A40 mainly via hydrogen-bonding. These results suggest that hydroxycinnamic acid and its structural analogues could inhibit A40 fibrillogenesis and the inhibition activity is enhanced with the increase of phenolic hydroxyl groups of inhibitors. These small molecules have huge potential to be developed into a novel aggregation inhibitor in neurodegenerative disorders.
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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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[
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.
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[
Nat Genet,
2002]
Mice that are homozygous with respect to a mutation (ax(J)) in the ataxia (ax) gene develop severe tremors by 2-3 weeks of age followed by hindlimb paralysis and death by 6-10 weeks of age. Here we show that ax encodes ubiquitin-specific protease 14 (Usp14). Ubiquitin proteases are a large family of cysteine proteases that specifically cleave ubiquitin conjugates. Although Usp14 can cleave a ubiquitin-tagged protein in vitro, it is unable to process polyubiquitin, which is believed to be associated with the protein aggregates seen in Parkinson disease, spinocerebellar ataxia type 1 (SCA1; ref. 4) and gracile axonal dystrophy (GAD). The physiological substrate of Usp14 may therefore contain a mono-ubiquitin side chain, the removal of which would regulate processes such as protein localization and protein activity. Expression of Usp14 is significantly altered in ax(J)/ax(J) mice as a result of the insertion of an intracisternal-A particle (IAP) into intron 5 of Usp14. In contrast to other neurodegenerative disorders such as Parkinson disease and SCA1 in humans and GAD in mice, neither ubiquitin-positive protein aggregates nor neuronal cell loss is detectable in the central nervous system (CNS) of ax(J) mice. Instead, ax(J) mice have defects in synaptic transmission in both the central and peripheral nervous systems. These results suggest that ubiquitin proteases are important in regulating synaptic activity in mammals.
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[
Development,
1991]
In wild-type Caenorhabditis elegans hermaphrodites, two bilaterally symmetric sex myoblasts (SMs) migrate anteriorly to flank the precise center of the gonad, where they divide to generate the muscles required for egg laying (J. E. Sulston and H. R. Horvitz (1977) Devl Biol. 56, 110-156). Although this migration is largely independent of the gonad, a signal from the gonad attracts the SMs to their precise final positions (J. H. Thomas, M. J. Stern and H. R. Horvitz (1990) Cell 62, 1041-1052). Here we show that mutations in either of two genes,
egl-15 and
egl-17, cause the premature termination of the migrations of the SMs. This incomplete migration is caused by the repulsion of the SMs by the same cells in the somatic gonad that are the source of the attractive signal in wild-type animals.