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.

Characterization and Biological Studies of the Terpenoids from Ganoderma resinaceum and Serpula similis (Agaricomycetes).

Mycochemical properties and bioactivities of Ganoderma resinaceum and Serpula similis remain unexplored. The present study assessed antioxidant, cytotoxicity, and cell migration abilities of Ganoderma and Serpula extracts, followed by their phytochemical analyses. The MTT assay was conducted to determine the cytotoxicity along with the cell migration studies in human cancer cell lines. The antioxidant profiles were evaluated through DPPH and FRAP assays. Furthermore, LC-MS/MS analysis was performed to elucidate the phytochemicals responsible for anticancer and antioxidant activities. Significant concentration-dependent cytotoxicities of 12.7% and 13.7% were observed against HCT 116 cell lines at 1% and 5% concentrations of the G. resinaceum extract, respectively. Similarly, significant concentration-dependent cytotoxicities of 6.7% and 25.5% were observed at 1% and 5% concentrations of the S. similis extract, respectively. The extracts of G. resinaceum and S. similis both shows better anti-migration potential in lung cancer cells. Both extracts demonstrated good scavenging activity on DPPH and ferric ion free radicals. LC-MS analysis revealed 11 compounds from S. similis and 15 compounds from G. resinaceum fruiting bodies. Compounds such as terpenoids, alkaloids, cytotoxic peptides, and other metabolites were identified as major components in both extracts. These extracts exhibited cytotoxic activity against HCT 116 cancer cells, along with moderate antioxidant activity. This implies that the extracts might be used as bioactive natural sources in the pharmaceutical and food industries.

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).

Cytoprotective effect exerted by geraniin in HepG2 cells is through microRNA mediated regulation of BACH-1 and HO-1.

Geraniin, a hydrolysable tannin, used in traditional medicine in Southeast Asia, is known to exhibit various biological activities. As an antioxidant it is known to up-regulate phase II enzyme Heme oxygenase-1 (HO-1). However its mechanism is not clearly understood. Nuclear factor erythroid-derived 2 related factor 2 (Nrf-2) is transcriptionally up-regulated by Extracellular signal-regulated kinase (ERK) 1/2 and retained in nucleus due to inactivated Glycogen synthase kinase 3 beta (GSK-3ß). Geraniin additionally down-regulates expression of microRNA 217 and 377 (miR-217 and miR-377) which target HO-1 mRNA. Expression of BTB and CNC homolog 1 (BACH-1), another regulator of HO-1, is also down-regulated by up-regulating microRNA 98 (miR-98), a negative regulator of BACH-1. Thus, geraniin up-regulates HO-1 expression both through activating its positive regulator Nrf-2 and by down-regulating its negative regulator BACH-1. Up-regulation of HO-1 also confers protection to HepG2 cells from tertiary butyl hydroperoxide (TBH) induced cytotoxicity. [BMB Reports 2017; 50(11): 560-565].

Protective effect of geraniin against carbon tetrachloride induced acute hepatotoxicity in Swiss albino mice.

Geraniin is a hydrolysable tannin, widely present in many plant species, specifically used in traditional medicines. It has been shown to exhibit strong antioxidant activity in vitro. This study was performed to investigate hepatoprotective activity of geraniin against carbon tetrachloride (CCl4) induced damage in Swiss albino mice. Mice were treated with 30 and 60 mg/kg geraniin for 10 days followed by CCl4 administration for 24 h. Increase in Serum biochemical marker enzymes and histological deteriorative changes of liver tissue after CCl4 administration were attenuated by geraniin. Geraniin significantly reduced CCl4 induced lipid peroxidation, increase in amount of glutathione, glutathione reductase and Heme oxygenase-1 (HO-1). On the other hand it inhibited significant reduction in catalase activity and expression caused by CCl4 administration. Pre-treatment with geraniin reduced phosphorylation of translation initiation factor eIF2α, at serine 51, caused by CCl4 exposure and reduced elevated expression of its upstream kinase, Heme-regulated Inhibitor (HRI). These results clearly demonstrate hepatoprotective activity of geraniin against CCl4-induced acute hepatotoxicity via its free radical scavenging and antioxidant activities.

SMAR1 binds to T(C/G) repeat and inhibits tumor progression by regulating miR-371-373 cluster.

Chromatin architecture and dynamics are regulated by various histone and non-histone proteins. The matrix attachment region binding proteins (MARBPs) play a central role in chromatin organization and function through numerous regulatory proteins. In the present study, we demonstrate that nuclear matrix protein SMAR1 orchestrates global gene regulation as determined by massively parallel ChIP-sequencing. The study revealed that SMAR1 binds to T(C/G) repeat and targets genes involved in diverse biological pathways. We observe that SMAR1 binds and targets distinctly different genes based on the availability of p53. Our data suggest that SMAR1 binds and regulates one of the imperative microRNA clusters in cancer and metastasis, miR-371-373. It negatively regulates miR-371-373 transcription as confirmed by SMAR1 overexpression and knockdown studies. Further, deletion studies indicate that a ~200 bp region in the miR-371-373 promoter is necessary for SMAR1 binding and transcriptional repression. Recruitment of HDAC1/mSin3A complex by SMAR1, concomitant with alteration of histone marks results in downregulation of the miRNA cluster. The regulation of miR-371-373 by SMAR1 inhibits breast cancer tumorigenesis and metastasis as determined by in vivo experiments. Overall, our study highlights the binding of SMAR1 to T(C/G) repeat and its role in cancer through miR-371-373.

Andrographolide protects liver cells from H2O2 induced cell death by upregulation of Nrf-2/HO-1 mediated via adenosine A2a receptor signalling.

BACKGROUND: Andrographolide, principle constituent of Andrographis paniculata Nees is used in traditional medicine in Southeast Asia and is known to exhibit various biological activities. Its antioxidant activity is due to its ability to activate one of the antioxidant enzymes, heme oxygenase-1 (HO-1) which is regulated transcriptionally through Nrf-2. However, molecular mechanism underlying activation of Nrf-2/HO-1 has not yet been clearly understood. METHODS: Protective effect of andrographolide against H2O2 induced cell death, reactive oxygen species and lipid peroxidation was observed in HepG2 cells. Ability of andrographolide to modulate G-protein coupled receptor (GPCR) mediated signalling was determined using in silico docking and gene expression was analyzed by qRT-PCR, confocal microscopy and western blot analysis. RESULTS: We clearly show that andrographolide via adenosine A2A receptor signalling leads to activation of p38 MAP kinase, resulting in upregulation of Nrf-2, its translocation to nucleus and activation of HO-1. Additionally, it activates adenylate cyclase resulting in cAMP formation which in turn activates protein kinase A leading to inhibition of GSK-3ß by phosphorylation. Inactivated GSK-3ß leads to retention of Nrf-2 in the nucleus leading to sustained expression of HO-1 by binding to its antioxidant response element (ARE). CONCLUSIONS: Thus, andrographolide probably by binding to adenosine A2a receptor activates Nrf-2 transcription and also inhibits its exclusion from the nucleus by inactivating GSK-3ß, together resulting in activation of HO-1. GENERAL SIGNIFICANCE: We speculate that andrographolide can be used as a therapeutic drug to combat oxidative stress implicated in pathogenesis of various diseases such as diabetes, osteoporosis, neurodegenerative diseases etc.

Sequence data mining in search of hookworm (Necator americanus) microRNAs.

The new world hookworm, Necator americanus is a soil-transmitted nematode responsible for Necatoriasis (a type of helminthiasis) in hosts such as humans, dogs, and cats. N. americanus genome and transcriptome has been sequenced and a draft assembly analysis has been published highlighting protein coding genes and possible drug target proteins. Hookworm microRNA identification, annotations and their public release is yet to be attempted. The same is evident from lack of hookworm miRNA information in related popular public nucleotide sequence repositories such as miRBase, GenBank, WormBase etc. Therefore, in the present study we addressed these issues using EST and assembled transcript sequence information of hookworm. Using computational approaches, we identified three miRNAs precursor sequences and their mature forms. We also identified their potential targets from hookworm ESTs and transcripts, and from human transcriptome. Overall, the results indicate presence of nematode specific miRNA homologs in N. americanus and shades light on their putative targets in worm itself and the human host.

Dose-dependent differential response of mammalian cells to cytoplasmic stress is mediated through the heme-regulated eIF2α kinase.

The heme-regulated inhibitor (HRI), a regulator of translation initiation, is known to be activated and upregulated, and it acts as either a cytoprotective player promoting cell survival or as an inducer of apoptosis during stress. However, the exact role of HRI in these two responses has not been elucidated. In the present investigation, using human cell lines, we attempted to unravel the molecular mechanism(s) of HRI-mediated differential response and the involved signaling pathways. While during low dose (5 µM) lead acetate treatment, cells did not show any diminished cell survival, significant level of apoptosis was observed at high dose (100 µM) lead acetate. Based on the results of an interactome analysis, we determined the interaction of HRI with PI-3-Kca, only at a low dose stress, which is followed by phosphorylation and activation of its downstream target, AKT. Interestingly, such an interaction and AKT activation was not observed at a high dose stress. On the other hand, an increased level of APAF-1 and activation of caspases were observed. These results indicate a critical role of HRI in cell survival during low dose stress, and in apoptosis at high dose stress. Furthermore, HRI knockdown cells are sensitized even to 5 µM lead treatment leading to caspase activation and apoptosis. Our results taken together thus elucidate for the first time the molecular mechanism and the involved signaling pathways for dose-dependent differential response of mammalian cells to lead exposure. These findings thus suggest the possibility of using HRI downregulation as a therapeutic strategy to sensitize cancer cells subjected to apoptogenic drugs.

miR-320a regulates erythroid differentiation through MAR binding protein SMAR1.

Erythropoiesis is controlled by a complex interplay of several signaling pathways and key transcription factors, as well as microRNAs (miRNAs). MicroRNAs function as critical modulators of gene expression for cellular processes. In the present study, we found that miR-320a inhibits erythroid differentiation by targeting Matrix Attachment Region binding protein SMAR1. miR-320a negatively regulates the expression of SMAR1 by directly binding to its 3'UTR. In response to mild DNA damage, miR-320a expression is decreased resulting in enhanced expression of SMAR1 protein, which in turn, reduces its targets, Bax and Puma inhibiting apoptosis. Our data demonstrate that during hemin-induced erythroid differentiation, enhanced expression of SMAR1 negatively correlates with miR-320a expression. Further analysis reveals that SMAR1 regulates erythroid differentiation, by binding to the promoter of miR-221/222, which play a crucial role in early erythropoiesis. Overall, our studies provide an insight into the regulation of hemin mediated erythroid differentiation of K562 cells through post-transcriptional regulation of SMAR1.

Chromatin remodelling protein SMAR1 inhibits p53 dependent transactivation by regulating acetyl transferase p300.

Acetylation of p53 is indispensable for its transcriptional activities and induction of apoptosis upon DNA damage. Here, we show that chromatin remodelling protein SMAR1 inhibits p53 acetylation and p53 dependent apoptosis by repressing p300 expression in response to DNA damage. The repression of p300 expression by SMAR1 is relieved upon treatment with proteosomal inhibitors MG132 and Lactacystin. We demonstrate that SMAR1 interacts with p53-p300 transcriptional complex and SMAR1 overexpression antagonizes p300 interaction with p53 and suppresses activation of p53 apoptotic targets and p53 regulated miRNA miR-34a. Conversely, knockdown of SMAR1 promotes p300 accumulation and p53 acetylation while ectopic expression of p300 rescues SMAR1 inhibition on p53. Collectively, these results indicate that SMAR1 is an important player in p300-p53 regulated DNA damage signalling pathway and can exert its effect on apoptosis in a transcription independent manner.

Coordinated regulation of p53 apoptotic targets BAX and PUMA by SMAR1 through an identical MAR element.

How tumour suppressor p53 bifurcates cell cycle arrest and apoptosis and executes these distinct pathways is not clearly understood. We show that BAX and PUMA promoters harbour an identical MAR element and are transcriptional targets of SMAR1. On mild DNA damage, SMAR1 selectively represses BAX and PUMA through binding to the MAR independently of inducing p53 deacetylation through HDAC1. This generates an anti-apoptotic response leading to cell cycle arrest. Importantly, knockdown of SMAR1 induces apoptosis, which is abrogated in the absence of p53. Conversely, apoptotic DNA damage results in increased size and number of promyelocytic leukaemia (PML) nuclear bodies with consequent sequestration of SMAR1. This facilitates p53 acetylation and restricts SMAR1 binding to BAX and PUMA MAR leading to apoptosis. Thus, our study establishes MAR as a damage responsive cis element and SMAR1-PML crosstalk as a switch that modulates the decision between cell cycle arrest and apoptosis in response to DNA damage.

Oxidative stress perturbs cell proliferation in human K562 cells by modulating protein synthesis and cell cycle.

Oxidative stress leads to perturbation of a variety of cellular processes resulting in inhibition of cell proliferation. This study has determined the effect of oxidative stress on protein synthesis in human K562 cells using a hydrophilic peroxyl radical initiator, AAPH and H(2)O(2). The results indicated that oxidative stress leads to a significant decrease in the rate of protein synthesis caused due to induced activation as well as expression of the erythroid cell-specific eIF-2alpha kinase, called the Heme Regulated Inhibitor (HRI). Elevated levels of HRI expression and activity were accompanied by increased lipid peroxidation and decreased cell proliferation. Further, oxidative stress also caused inactivation of p34(cdc2) kinase, thereby arresting cell division leading to apoptosis. Thus, the data provides the mechanism of inhibition of protein synthesis and perturbation of a cell cycle regulatory protein leading to inhibition of cell proliferation in K562 cells during oxidative stress.