Earthworm Lumbricus rubellus MT-2 : metal binding and protein folding of a true Cadmium-MT

Earthworms express, as most animals, metallothioneins (MTs)—small, cysteine-rich proteins that bind d10 metal ions (Zn(II), Cd(II), or Cu(I)) in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II) and Zn(II). Crucially, whilst a single Cd7wMT-2 species was isolated from wMT-2-expressing E. coli cultures supplemented with Cd(II), expressions in the presence of Zn(II) yielded mixtures. The average affinities of wMT-2 determined for either Cd(II) or Zn(II) are both within normal ranges for MTs; hence, differential behaviour cannot be explained on the basis of overall affinity. Therefore, the protein folding properties of Cd- and Zn-wMT-2 were compared by 1H NMR spectroscopy. This comparison revealed that the protein fold is better defined in the presence of cadmium than in the presence of zinc. These differences in folding and dynamics may be at the root of the differential behaviour of the cadmium- and zinc-bound protein in vitro, and may ultimately also help in distinguishing zinc and cadmium in the earthworm in vivo

The nematode Caenorhabditis elegans, stress and aging:Identifying the complex interplay of genetic pathways following the treatment with humic substances

Low concentrations of the dissolved leonardite humic acid HuminFeed(®) (HF) prolonged the lifespan and enhanced the thermal stress resistance of the model organism Caenorhabditis elegans. However, growth was impaired and reproduction delayed, effects which have also been identified in response to other polyphenolic monomers, including Tannic acid, Rosmarinic acid, and Caffeic acid. Moreover, a chemical modification of HF, which increases its phenolic/quinonoid moieties, magnified the biological impact on C. elegans. To gain a deep insight into the molecular basis of these effects, we performed global transcriptomics on young adult (3 days) and old adult (11 days) nematodes exposed to two different concentrations of HF. We also studied several C. elegans mutant strains in respect to HF derived longevity and compared all results with data obtained for the chemically modified HF. The gene expression pattern of young HF-treated nematodes displayed a significant overlap to other conditions known to provoke longevity, including various plant polyphenol monomers. Besides the regulation of parts of the metabolism, transforming growth factor-beta signaling, and Insulin-like signaling, lysosomal activities seem to contribute most to HF’s and modified HF’s lifespan prolonging action. These results support the notion that the phenolic/quinonoid moieties of humic substances are major building blocks that drive the physiological effects observed in C. elegans

The significance of genome-wide transcriptional regulation in the evolution of stress tolerance.

It is widely recognized that stress plays an important role in directing the adaptive adjustment of an organism to changing environments. However, very little is known about the evolution of mechanisms that promote stress-induced variation. Adaptive transcriptional responses have been implicated in the evolution of tolerance to natural and anthropogenic stressors in the environment. Recent technological advances in transcriptomics provide a mechanistic understanding of biological pathways or processes involved in stress-induced phenotypic change. Furthermore, these studies are (semi) quantitative and provide insight into the reaction norms of identified target genes in response to specific stressors. We argue that plasticity in gene expression reaction norms may be important in the evolution of stress tolerance and adaptation to environmental stress. This review highlights the consequences of transcriptional plasticity of stress responses within a single generation and concludes that gene promoters containing a TATA box are more capable of rapid and variable responses than TATA-less genes. In addition, the consequences of plastic transcriptional responses to stress over multiple generations are discussed. Based on examples from the literature, we show that constitutive over expression of specific stress response genes results in stress adapted phenotypes. However, organisms with an innate capacity to buffer stress display plastic transcriptional responses. Finally, we call for an improved integration of the concept of phenotypic plasticity with studies that focus on the regulation of transcription. © Springer Science+Business Media B.V. 2010

Metallothionein 2 and Heat Shock Protein 72 Protect Allolobophora chlorotica from Cadmium But Not Nickel or Copper Exposure:Body Malformation and Coelomocyte Functioning

Earthworms serve as good indicators of heavy metal contamination due to their innate sensitivity towards soil pollution. However, to date, not many studies have focused on endogeic earthworms, such as the omnipresent Allolobophora chlorotica. The current study was designed to verify whether this earthworm could serve as a novel distinctively susceptible species for environmental contamination studies. We show that the dermal exposure to Cu, Ni, and Cd affected the mortality and morphology of A. chlorotica, and the number and functioning of coelomocytes. These features particularly were pronounced in animals treated with Ni and Cu and interestingly to a lesser extend with Cd. In contrast, Cd induced a strong expression of metallothioneins (MT-2) and heat shock proteins (HSP72). The presence of MT-2 was detected not only in coelomocytes but also in the intestine, blood vessels, and epidermis. In conclusion, Allolobophora chlorotica coelomocytes are adopted to respond differentially to various heavy metals, generating powerful response towards potentially most dangerous exogenous non-essential elements

Studying MT regulation in L. rubellus, the red earthworm, suggests novel activation mechanisms

Metallothioneins (MTs) are multifunctional proteins which are of major importance in a variety of stress related cellular responses. The majority of studies on the transcriptional regulation of MTs have, to date, been performed in vertebrate species and have revealed a common activation mechanism, namely the binding of the metal transcription factor 1 (MTF-1) to metal responsive elements (MREs) located on the promotor region of MT genes. This present study provides first insights into the regulation of earthworm MTs by means of L. rubellus and the Cd-inducible wMT2 isoform. Electric mobility shift assay (EMSA) was applied to investigate whether MREs are functional binding sites and therefore involved in basal and Cd-induced wMT2 activation. DNase I Footprinting was used to evaluate putative protein binding sites other than MREs on the wMT2 promotor region. EMSA (encompassing three probes, each including one of the MRE binding sites of the wMT2 promotor region) uncovered the presence of a Zn-dependent shift in the cytosolic protein fraction which was independent of sample origin (including control and Cd-exposed earthworms). Subsequent DNase I Footprinting revealed that the MRE flanking regions (rather than the MREs themselves) are the putative transcriptional activator sites. Moreover, the latter method identified further functional transcription factor binding regions. These results indicate that the mechanisms of MTF-1 binding to MREs to activate MT transcription in vertebrates might not be universally applicable to invertebrates and would confirm the fact that neither the L. rubellus nor the nematode C. elegans genome contains MTF-1 homologs. Ongoing experiments aim to identify the transcription factors involved in basal and/or metal-induced transcriptional activation of the earthworm wMT2 isoform. Revealing the transcriptional activation mechanism of the earthworm MT will advance and update invertebrate MT research and improve our understanding of metal detoxification processes

The double mutation of cytochrome P450's and fatty acid desaturases affect lipid regulation and longevity in C. elegans

AbstractAn imbalance between energy uptake and energy expenditure can lead to obesity and increase the risk of coronary heart disease, high blood pressure, stroke, type II diabetes and some cancers. Given that key elements of the energy pathway are evolutionary conserved, invertebrate research is an attractive alternative that overcomes the many legislative, financial and experimental hurdles typical of research with higher metazoan animals. Recent studies have suggested that some members of the cytochrome P450 superfamily are involved in lipid metabolism in addition to the traditional xenobiotic activity. To investigate this notion in more detail, the present study aimed to pinpoint phenotypic, genetic and genomic-level responses of Caenorhabditis elegans using selected deletion mutants including fat-5 (a member of the Δ9 desaturases) and cyp-35A2 (a member of the cytochrome P450 family). The creation of a fat-5(tm420);cyp-35A2(gk317) mutant uncovered that the deletion of both genes resulted in a strain which is marked by an extended lifespan. Furthermore, it diminished the overall level of Nile Red positive compartments, which is indicative of a change in lipid metabolism. Comprehensive transcriptomics revealed that several genes involved in aging and lipid transport/homeostasis were modulated following the double deletion of fat-5 and cyp-35A2. Taken together, the results suggest the presence of a putative correlation between longevity and lipid regulation and given that both genes have human homologs, this finding may offer a new lead to investigate in higher organisms

The Metabolomic Responses of Caenorhabditis elegans to Cadmium Are Largely Independent of Metallothionein Status, but Dominated by Changes in Cystathionine and Phytochelatins

Cadmium is a widely distributed toxic environmental pollutant. Using proton NMR spectroscopy and UPLC-MS, we obtained metabolic profiles from the model organism Caenorhabditis elegans exposed to sublethal concentrations of cadmium. Neither in the presence nor absence of cadmium did the metallothionein status (single or double mtl knockouts) markedly modulate the metabolic profile. However, independent of strain, cadmium exposure resulted in a decrease in cystathionine concentrations and an increase in the nonribosomally synthesized peptides phytochelatin-2 and phytochelatin-3. This suggests that a primary response to low levels of cadmium is the differential regulation of the C. elegans trans-sulfuration pathway, which channels the flux from methionine through cysteine into phytochelatin synthesis. These results were backed up by the finding that phytochelatin synthase mutants (pcs-1) were at least an order of magnitude more sensitive to cadmium than single or double metallothionein mutants. However, an additive sensitivity toward cadmium was observed in the mtl-1; mtl-2; pcs-1 triple mutant

Nutritive Manganese and Zinc Overdosing in Aging C. elegans Result in a Metallothionein‐Mediated Alteration in Metal Homeostasis

Scope Manganese (Mn) and zinc (Zn) are not only essential trace elements, but also potential exogenous risk factors for various diseases. Since the disturbed homeostasis of single metals can result in detrimental health effects, concerns have emerged regarding the consequences of excessive exposures to multiple metals, either via nutritional supplementation or parenteral nutrition. This study focuses on Mn‐Zn‐interactions in the nematode Caenorhabditis elegans (C. elegans) model, taking into account aspects related to aging and age‐dependent neurodegeneration. Methods and Results Chronic co‐exposure of C. elegans to Mn and Zn increases metal uptake, exceeding levels of single metal exposures. Supplementation with Mn and/or Zn also leads to an age‐dependent increase in metal content, a decline in overall mRNA expression, and metal co‐supplementation induced expression of target genes involved in Mn and Zn homeostasis, in particular metallothionein 1 (mtl‐1). Studies in transgenic worms reveal that mtl‐1 played a prominent role in mediating age‐ and diet‐dependent alterations in metal homeostasis. Metal dyshomeostasis is further induced in parkin‐deficient nematodes (Parkinson's disease (PD) model), but this did not accelerate the age‐dependent dopaminergic neurodegeneration. Conclusions A nutritive overdose of Mn and Zn can alter interactions between essential metals in an aging organism, and metallothionein 1 acts as a potential protective modulator in regulating homeostasis.DFG, 316442145, FOR 2558: Interaktionen von essenziellen Spurenelementen in gesunden und erkrankten älteren Menschen(TraceAge