Identification and characterization of multidomain monothiol glutaredoxin 3 from diploblastic Hydra.

Intracellular antioxidant glutaredoxin controls cell proliferation and survival. Based on the active site, structure, and conserved domain motifs, it is classified into two classes. Class I contains dithiol Grxs with two cysteines in the consensus active site sequence CXXC, while class II has monothiol Grxs with one cysteine residue in the active site. Monothiol Grxs can also have an additional N-terminal thioredoxin (Trx)-like domain. Previously, we reported the characterization of Grx1 from Hydra vulgaris (HvGrx1), which is a dithiol isoform. Here, we report the molecular cloning, expression, analysis, and characterization of another isoform of Grx, which is the multidomain monothiol glutaredoxin-3 from Hydra vulgaris (HvGrx3). It encodes a protein with 303 amino acids and is significantly larger and more divergent than HvGrx1. In-silico analysis revealed that Grx1 and Grx3 have 22.5% and 9.9% identical nucleotide and amino acid sequences, respectively. HvGrx3 has two glutaredoxin domains and a thioredoxin-like domain at its amino terminus, unlike HvGrx1, which has a single glutaredoxin domain. Like other monothiol glutaredoxins, HvGrx3 failed to reduce glutathione-hydroxyethyl disulfide. In the whole Hydra, HvGrx3 was found to be expressed all over the body column, and treatment with H2O2 led to a significant upregulation of HvGrx3. When transfected in HCT116 (human colon cancer cells) cells, HvGrx3 enhanced cell proliferation and migration, indicating that this isoform could be involved in these cellular functions. These transfected cells also tolerate oxidative stress better.

Glutaredoxin 1 from Evolutionary Ancient Hydra: Characteristics of the Enzyme and Its Possible Functions in Cell.

Glutaredoxin (Grx) is an antioxidant redox protein that uses glutathione (GSH) as an electron donor. Grx plays a crucial role in various cellular processes, such as antioxidant defense, control of cellular redox state, redox control of transcription, reversible S-glutathionylation of specific proteins, apoptosis, cell differentiation, etc. In the current study, we have isolated and characterized dithiol glutaredoxin from Hydra vulgaris Ind-Pune (HvGrx1). Sequence analysis showed that HvGrx1 belongs to the Grx family with the classical Grx motif (CPYC). Phylogenetic analysis and homology modeling revealed that HvGrx1 is closely related to Grx2 from zebrafish. HvGrx1 gene was cloned and expressed in Escherichia coli cells; the purified protein had a molecular weight of 11.82 kDa. HvGrx1 efficiently reduced ß-hydroxyethyl disulfide (HED) with the temperature optimum of 25°C and pH optimum 8.0. HvGrx1 was ubiquitously expressed in all body parts of Hydra. Expression of HvGrx1 mRNA and enzymatic activity of HvGrx1 were significantly upregulated post H2O2 treatment. When expressed in human cells, HvGrx1 protected the cells from oxidative stress and enhanced cell proliferation and migration. Although Hydra is a simple invertebrate, HvGrx1 is evolutionary closer to its homologs from higher vertebrates (similar to many other Hydra proteins).

A novel thioredoxin glutathione reductase from evolutionary ancient metazoan Hydra.

Thioredoxin (Trx) and glutathione disulfide (GSSG), are regenerated in reduced state by thioredoxin reductase (TrxR) and glutathione reductase (GR) respectively. A novel protein thioredoxin glutathione reductase (TGR) capable of reducing Trx as well as GSSG, linking two redox systems, has only been reported so far from parasitic flat worms and mammals. For the first time, we report a multifunctional antioxidant enzyme TGR from the nonparasitic, nonmammalian cnidarian Hydra vulgaris (HvTGR) which is a selenoprotein with unusual fusion of a TrxR domain with glutaredoxin (Grx) domain. We have cloned and sequenced HvTGR which encodes a polypeptide of 73 kDa. It contains conserved sequence CPYC of Grx domain, and CVNVGC and GCUG domains of thioredoxin reductase. Phylogenetic analysis revealed HvTGR to be closer to TGR from mammals rather than to TGR from parasitic helminths. We then subcloned HvTGR in plasmid pSelExpress-1 and expressed it in HEK293T cells to ensure selenocysteine incorporation. Purified HvTGR showed Grx, glutathione reductase and TrxR activities. Both thioredoxin and GSSG disulfide reductase activities were inhibited by 1-Chloro-2,4-dinitrobenzene (DNCB) supporting the existence of an essential selenocysteine residue. HvTGR expression was induced in response to H2O2 in Hydra. Interestingly, inhibition of HvTGR by DNCB, inhibited regeneration in Hydra indicating its involvement in other cellular processes.

Cloning and characterization of Thioredoxin 1 from the Cnidarian Hydra.

Thioredoxins, small disulphide-containing redox proteins, play an important role in the regulation of cellular thiol redox balance through their disulfide reductase activity. In this study, we have identified, cloned, purified and characterized thioredoxin 1 (HvTrx1) from the Cnidarian Hydra vulgaris Ind-Pune. Bioinformatics analysis revealed that HvTrx1 contains an evolutionarily conserved catalytic active site Cys-Gly-Pro-Cys and shows a closer phylogenetic relationship with vertebrate Trx1. Optimum pH and temperature for enzyme activity of purified HvTrx1 was found to be pH 7.0 and 25°C, respectively. Enzyme activity decreased significantly at acidic or alkaline pH as well as at higher temperatures. HvTrx1 was found to be expressed ubiquitously in whole mount in situ hybridization. Treatment of Hydra with hydrogen peroxide (H2O2), a highly reactive oxidizing agent, led to a significant increase in gene expression and enzyme activity of Trx1. Further experiments using PX12, an inhibitor of Trx1, indicated that Trx1 plays an important role in regeneration in Hydra. Finally, by using growth assay in Escherichia coli and wound healing assay in human colon cancer cells, we demonstrate that HvTrx1 is functionally active in both prokaryotic and eukaryotic heterologous systems.

Excess hydrogen peroxide inhibits head and foot regeneration in hydra by affecting DNA repair and expression of essential genes.

Reactive oxygen species (ROS) are necessary for various cellular processes. However, excess ROS cause damage to many biological molecules and therefore must be tightly regulated in time and space. Hydrogen peroxide (H2 O2 ) is the most commonly used ROS as second messenger in the cell. It is a relatively long-lived freely diffusible signaling molecule during early events of injury. In the Cnidarian hydra, injury-induced ROS production is essential for regeneration to proceed. In the present study, we have examined influence of varying exposure to H2 O2 on head and foot regeneration in the middlepieces of trisected hydra. We find that longer (4 hours) exposure to 1 mM H2 O2 inhibits both head and foot regeneration while shorter exposure (2 hours) does not. Longer exposure to H2 O2 resulted in extensive damage to DNA that could not be repaired, probably due to suboptimal induction of APE1, an enzyme necessary for base excision repair (BER). Concomitantly, genes involved in activation of Wnt pathway, necessary for head regeneration, were significantly downregulated. This appeared to be due to failure of both stabilization and transient nuclear localization of ß-catenin. Similarly, genes involved in foot regeneration were also downregulated on longer exposure to H2 O2 . Thus, exposure to excess ROS inhibits regenerative processes in hydra through reduced expression of genes involved in regeneration and diminished DNA repair.

DNA repair enzyme APE1 from evolutionarily ancient Hydra reveals redox activity exclusively found in mammalian APE1.

Only mammalian apurinic/apyrimidinic endonuclease1 (APE1) has been reported to possess both DNA repair and redox activities. C terminal of the protein is required for base excision repair, while the redox activity resides in the N terminal due to cysteine residues at specific positions. APE1s from other organisms studied so far lack the redox activity in spite of having the N terminal domain. We find that APE1 from the Cnidarian Hydra exhibits both endonuclease and redox activities similar to mammalian APE1. We further show the presence of the three indispensable cysteines in Hydra APE1 for redox activity by site directed mutagenesis. Importance of redox domain but not the repair domain of APE1 in regeneration has been demonstrated by using domain-specific inhibitors. Our findings clearly demonstrate that the redox function of APE1 evolved very early in metazoan evolution and is not a recent acquisition in mammalian APE1 as believed so far.