EIF4A3 targeted therapeutic intervention in glioblastoma multiforme using phytochemicals from Indian medicinal plants - an integration of phytotherapy into precision onco-medicine.

Glioblastoma Multiforme (GBM), an aggressive brain tumor (grade-IV astrocytoma), poses treatment challenges. Poor prognosis results from the rapid growth, highlighting the role of EIF4A3 in regulating non-coding RNAs. EIF4A3 promotes the expression of several non-coding RNAs, viz, Circ matrix metallopeptidase 9 (MMP9), a prominent oncogene, by interacting with the upstream region of the circMMP9 mRNA transcript and acts on cell proliferation, migration, and invasion of GBM. However, research shows that EIF4A3 knockdown inhibits glioblastoma progression and increases apoptosis. In this study, we explored the efficiency of the phytochemicals from plants like Withania somnifera and Castanea sativa with potential anti-glioblastoma effects as obtained from the Indian Medicinal Plants, Phytochemistry and Therapeutics (IMPPAT) database. Consequently, we have performed a virtual screening of the compounds against the protein EIF4A3. We further investigated the efficiency of the shortlisted compounds based on docking scores evaluated using GOLD, AutoDock4.2, LeDock, and binding free energy analyses using Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA). Among the phytochemicals studied so far, several Withania-specific compounds from Withania somnifera and a single dietary compound, viz., Thiamine from Castanea sativa, have exhibited comparatively good blood-brain barrier permeability, significant binding affinity towards the EIF4A3, and good ADMET properties. Furthermore, we have verified the interaction stability of the lead molecules with EIF4A3 using MD simulations. Thus, the present study offers an opportunity to develop drug candidates targeting glioblastoma caused by EIF4A3 over-expression, integrating phytotherapy into precision oncology to create tailored and precise natural treatment strategies for cancer.Communicated by Ramaswamy H. Sarma.

Precision medicine advancements in glioblastoma: A systematic review.

Background: Glioblastoma multiforme, commonly known as GBM or glioblastoma is a grade IV astrocytoma. Brain tumors are difficult to treat and lead to poor prognosis and survival in patients. Gliomas are categorized into four different grades among which GBM is the worst grade primary brain tumor with a survival of less than a year. The genomic heterogeneity of the brain tumor results in different profiles for patients diagnosed with glioblastoma. Precision medicine focuses on this specific tumor type and suggests specialized treatment for better prognosis and overall survival (OS). Purpose: With the recent advancements in Genome-Wide Studies (GWS) and various characterizations of brain tumors based on genetic, transcriptomic, proteomic, epigenetic, and metabolomics, this review discusses the advancements and opportunities of precision medicine therapeutics, drugs, and diagnosis methods based on the different profiles of glioblastoma. Methods: This review has exhaustively surveyed several pieces of works from various literature databases. Conclusion: It is evident that most primary brain tumors including glioblastoma require specific and precision therapeutics for better prognosis and OS. In present and future, molecular understanding and discovering specific therapies are essential for treatment in the field of neurooncology.

Quercetin Attenuates Copper-Induced Apoptotic Cell Death and Endoplasmic Reticulum Stress in SH-SY5Y Cells by Autophagic Modulation.

An increase in anthropogenic activities results in metal contamination in the ecosystem which has proven to be a major health risk in humans, as they make entry into cellular organelles via agricultural products. Copper (Cu) is one such metal that acts as an essential cofactor for the activity of several enzymes, one being the cytochrome c oxidase. The increasing number of evidence suggests a substantial correlation of Cu overload with neurodegenerative disorders, including Parkinson's disease (PD). We aim to explore quercetin, a well-known polyphenol, as an alternative for combating Cu-induced toxicity in human neuroblastoma SH-SY5Y secondary cell lines. We observed that Cu increased intracellular reactive oxygen species (ROS) levels, triggered morphological deformities and condensation of nuclei, caused an imbalance in the mitochondrial membrane potential (MMP), and finally induced apoptotic cell deaths. We further investigated the effects of Cu in modulating the pro- and anti-apoptotic proteins, such as Bax, Bcl-2, etc. However, quercetin reversed these changes owing to its antioxidant and anti-apoptotic properties, resulting in autophagy induction as an outcome of upregulation of autophagosome-bound microtubules-associated protein light chain-3 (LC3II). Besides, we investigated the role of Cu in stimulating ER stress proteins, viz. PERK, CHOP, and the concomitant responses of quercetin in restoring the ER homeostasis in cellular organelles like mitochondria and ER, against Cu-induced toxic insults by modulating autophagic pathways. Overall, this research work proposes a remedial approach for Cu-mediated neurotoxicity through understanding the diverse molecular signaling inside a cell with an aim to develop effective therapeutics.

Reduced Graphene Oxide Nanosheets for Selective Picomolar Detection of Bovine Serum Albumin.

In this paper, ultra-low level selective detection of bovine serum albumin (BSA) has been demonstrated, based on chemically derived graphene i.e., reduced graphene oxide (RGO) nanosheets. The working principle of the sensor is based upon change in conductance of the RGO nanosheets with different concentration of BSA. The change in conductance is based on the charge transfer between BSA and functional groups of RGO. The morphological and structural characterizations of RGO nanosheets were carried out by scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). Raman spectroscopy is performed to further validate the interaction between RGO sensing layer and BSA molecules. Electrical impedance spectroscopy is performed to observe the impedance variation when BSA interacts with RGO. The sensor device exhibits sensitivity of 10 nA/pM. The lower limit of detection (LOD) of the sensor is found to be 1 pM and response time around 35 s, confirming very high sensitivity for BSA. All electrical (current-voltage) measurements were carried out at 2 V bias for low power operation. The sensor exhibits highest sensitivity at 30 °C and for RGO thickness ~4 nm. The RGO based sensor device is selective towards BSA when compared to proteins like L-Histidine, HSA, BHB and Biotin. Our results suggest that RGO based devices are promising for low-cost, portable and real time detection of BSA at room temperature.

Multi-target inhibition ability of neohesperidin dictates its neuroprotective activity: Implication in Alzheimer's disease therapeutics.

The polygenic nature of Alzheimer's disease (AD) and cross-talk between several signaling cascades make it harder to decode the disease pathogenesis. ß-secretase (BACE1) works upstream in the amyloidogenic processing of amyloid precursor protein (APP) to generate Aß that rapidly aggregates to form fibrils, the most abundant component of plaques observed in AD brains. Here, we report dual inhibition of BACE1 and Aß aggregation by neohesperidin, a flavonoid glycoconjugate, using multi-spectroscopic approaches, force microscopy, molecular modeling, and validated the potency in SH-SY5Y neuroblastoma cell lines. Steady-state and time-resolved fluorescence reveal that neohesperidin binds close to the catalytic aspartate dyad. This binding conformationally restricts the protein in closed form which possibly precludes APP recognition and thereby inhibits BACE1 activity. Neohesperidin also dose-dependently inhibits the amyloid fibril formation, as evident from ANS, ThT assay, and AFM. Neohesperidin ameliorates aggregated Aß25-35 induced ROS generation and mitochondrial dysfunction in the SH-SY5Y cell line. As a result, the amyloid induced apoptosis is significantly prohibited and normal neuronal morphology is rescued. These findings suggest neohesperidin as an inhibitor of the pathogenic conversion of Aß to fibrillar amyloid assembly. Neohesperidin thus emerges as a non-toxic multi-potent scaffold for the development of AD therapeutics.

Neuroprotective Role of Quercetin on Rotenone-Induced Toxicity in SH-SY5Y Cell Line Through Modulation of Apoptotic and Autophagic Pathways.

The detrimental impact on the food chain due to the overuse of rotenone is partly responsible for alpha-synuclein (α-syn) mediated neurotoxicity. It is hypothesized that rotenone overdose leads to cytosolic proteopathy resulting in modulation of apoptosis and autophagic pathways. The aim of our study is to explore the neuroprotective role of quercetin, a beneficial polyphenol against rotenone-induced neurotoxicity in dopaminergic human SH-SY5Y cell lines. In our study we demonstrated the correlation of rotenone-induced neurotoxicity through elevation of intracellular reactive oxygen species (ROS) and imbalance in the mitochondrial membrane potential (MMP). Moreover, the morphological distortion of cell, condensation of nuclei, externalization of the inner phosphatidylserine, cleavage of caspase 3, and Poly ADP Ribose Polymerase (PARP) confirmed apoptosis. However, all these lethal effects were ameliorated by treatment of quercetin to the cells. On the other hand rotenone has a strong effect on autophagy which is a regulated degrading and recycling cellular process to remove dysfunctional proteins. Indeed, rotenone-mediated autophagy resulted in the enhancement of autophagosome-bound microtubule-associated protein light chain-3 (LC3-II) expression. Furthermore, excess accumulation of acidic vesicles was detected in presence of rotenone. Lysosome associated membrane protein (LAMP-2A) is yet another crucial protein that recruits overexpressed or misfolded proteins into the lumen of lysosome to trigger autophagy. In all cases the impact of rotenone on the cells acquired significant protection through quercetin treatment. In the present work we therefore opine the prospects of quercetin as a therapeutic candidate against neurotoxicity.

Iron chelator Deferoxamine protects human neuroblastoma cell line SH-SY5Y from 6-Hydroxydopamine-induced apoptosis and autophagy dysfunction.

BACKGROUND: Intracellular iron involves in Fenton's reaction-mediated Hydroxyl radical (OH·) generation by reacting with the neurotoxic agent 6-Hydroxydopamine (6-OHDA) autoxidation derivative Hydrogen Peroxide (H2O2). Several studies have been conducted so far on the neuroprotective activities of the iron chelator Deferoxamine (DFO) but little or no clear evidence about the underlying cellular mechanism is available. METHODS: The present study was conducted on Human neuroblastoma cell line SH-SY5Y in the absence or presence of 6-OHDA or H2O2 and / or DFO. Following incubation, cell viability assay, intracellular reactive oxygen species (ROS) determination, flow cytometric quantification of apoptotic cells followed by nuclear staining, intracellular tracking of transfected fusion construct of microtubule-associated protein 1B-light chain with Green fluorescent protein - Red fluorescent protein (LC3B-GFP-RFP reporters) and immunocytochemistry of intracellular Cathepsin protein by confocal microscopy, were conducted. In addition, western blotting was carried out to detect expressions of apoptotic and autophagy related proteins. RESULTS: This study confirmed the neuroprotective potential of DFO by inhibiting 6-OHDA-mediated cell death and ROS generation. Reduced percentage of apoptotic cells and appearance of altered nuclei architecture followed by a reduced expression of cleaved PARP (Poly-ADP-ribose Polymerase) and cleaved Caspase-3 were observed upon DFO treatment against 6-OHDA, and as well as against H2O2 in SH-SY5Y cell lines. Besides, DFO induced the intracellular autophagolysosome formation (red puncta) rather than autophagosome (yellow puncta) only. Thereafter it was observed that DFO restored the expression of intracellular lysosomal protease Cathepsin and reduced the expression of the LC3-II. CONCLUSION: Taken together, this study clearly demonstrated that the anti-Fenton activity of DFO inhibited apoptosis and caused blockade in ALP or autophagy dysfunction in SH-SY5Y cell lines. These outcomes further suggest that DFO provides neuroprotection by inhibiting apoptosis and inducing the progression of Autophagy- lysosomal pathway (ALP).

Iron-Induced Apoptotic Cell Death and Autophagy Dysfunction in Human Neuroblastoma Cell Line SH-SY5Y.

Iron accumulation plays a major role in neuronal cell death which has severe effects on mental health like neurodegenerative disorders. The present work aims to explore the involvement of molecular pathways involved in iron-mediated neuronal cell death using Ferric Ammonium Citrate (FAC) as a source of iron to treat neuroblastoma SH-SY5Y cells. In this study, it was found that cytotoxicity induced by iron treatment is highly correlated with enhanced intracellular reactive oxygen species (ROS) generation and loss of mitochondrial integrity. Appearance of early and late apoptotic cells with altered nuclear morphology and increased expression of effector proteins, i.e., cleaved Caspase 3 and cleaved PARP (Poly-ADP-ribose Polymerase), clearly confirmed iron-induced apoptotic cell deaths. Furthermore, excess accumulation of acidic vesicles and microtubule-associated protein 1 light chain 3 (LC3) puncta and LC3II/I expressions were observed. Simultaneously, ultrastructural studies of SH-SY5Y cells demonstrated the accumulation of a large number of autophagosomes, autophagic vacuolization, and swollen mitochondria which further confirmed the induction of autophagy concomitant with mitochondrial damage. Furthermore, increased incorporation of lysosome-specific dye, LysoTracker Deep Red, and the red fluorescence retention of LC3-GFP-RFP constructs indicates the incomplete autophagy or autophagy dysfunction due to altered lysosomal activity. Hence, the present work unveiled the interruption in autophagy progression caused by the plausible suppression of lysosomal activity due to iron treatment resulting in autophagic cell death in SH-SY5Y cell lines. In general, both apoptotic and autophagic pathways were prominent and each of the pathways played their prospective roles, in iron-mediated neuronal cell death.

Differential antibacterial response exhibited by graphene nanosheets toward gram-positive bacterium Staphylococcus aureus.

Two different morphological forms of graphene nanosheets: improved reduced graphene oxide (IRGO) and modified reduced GO (rGO) (MRGO) have been synthesised by improved and modified methods, respectively. Physical characterisations of these graphene nanosheets were carried out using X-ray diffraction, Fourier transform infrared spectroscopy, and Raman spectroscopy. Colloidal stability of these nanosheets toward a selected bacterium (e.g. Staphylococcus aureus) was ascertained by zeta potential. In the present study, the authors for the first time made an attempt to study and compare the potentialities of these two different forms of graphene nanosheets as efficient bactericidal agents. Field-emission scanning electron microscopy and TEM with energy dispersive X-ray spectroscopy (EDAX) studies of IRGO and MRGO have been carried out to explore their underlying mechanism of antibacterial responses through physical as well as chemical interactions with the selected bacterial species.

Phyto-assisted synthesis of bio-functionalised silver nanoparticles and their potential anti-oxidant, anti-microbial and wound healing activities.

Bio- synthesis of silver nanoparticles (AgNPs) was made by using the aqueous leaf extract of Ardisia solanacea. Rapid formation of AgNPs was observed from silver nitrate upon treatment with the aqueous extract of A. solanacea leaf. The formation and stability of the AgNPs in the colloidal solution were monitored by UV-visible spectrophotometer. The mean particle diameter of AgNPs was calculated from the DLS with an average size ∼4 nm and ∼65 nm. ATR-FTIR spectroscopy confirmed the presence of alcohols, aldehydes, flavonoids, phenols and nitro compounds in the leaf which act as the stabilizing agent. Antimicrobial activity of the synthesized AgNPs was performed using agar well diffusion and broth dilution method against the Gram-positive and Gram-negative bacteria. Further, robust anti-oxidative potential was evaluated by DPPH assay. The highest antimicrobial activity of synthesized AgNPs was found against Pseudomonas aeruginosa (28.2 ± 0.52 mm) whereas moderate activity was found against Bacillus subtilis (16.1 ± 0.76), Candida kruseii (13.0 ± 1.0), and Trichophyton mentagrophytes (12.6 ± 1.52). Moreover, the potential wound healing activity was observed against the BJ-5Ta normal fibroblast cell line. Current research revealed that A. solanacea was found to be a suitable source for the green synthesis of silver nanoparticles.

Biogenic synthesis of silver nanoparticles from Cassia fistula (Linn.): In vitro assessment of their antioxidant, antimicrobial and cytotoxic activities.

The present study reports on biogenic-synthesised silver nanoparticles (AgNPs) derived by treating Ag ions with an extract of Cassia fistula leaf, a popular Indian medicinal plant found in natural habitation. The progress of biogenic synthesis was monitored time to time using a ultraviolet-visible spectroscopy. The effect of phytochemicals present in C. fistula including flavonoids, tannins, phenolic compounds and alkaloids on the homogeneous growth of AgNPs was investigated by Fourier-transform infrared spectroscopy. The dynamic light scattering studies have revealed an average size and surface Zeta potential of the NPs as, -39.5 nm and -21.6 mV, respectively. The potential antibacterial and antifungal activities of the AgNPs were evaluated against Bacillus subtilis, Staphylococcus aureus, Candida kruseii and Trichophyton mentagrophytes. Moreover, their strong antioxidant capability was determined by radical scavenging methods (1,1-diphenyl-2-picryl-hydrazil assay). Furthermore, the AgNPs displayed an effective cytotoxicity against A-431 skin cancer cell line by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, with the inhibitory concentration (IC50) predicted as, 92.2 ± 1.2 µg/ml. The biogenically derived AgNPs could find immense scope as antimicrobial, antioxidant and anticancer agents apart from their potential use in chemical sensors and translational medicine.

Multi-target screening mines hesperidin as a multi-potent inhibitor: Implication in Alzheimer's disease therapeutics.

Alzheimer's disease (AD) is the most frequent form of neurodegenerative disorder in elderly people. Involvement of several pathogenic events and their interconnections make this disease a complex disorder. Therefore, designing compounds that can inhibit multiple toxic pathways is the most attractive therapeutic strategy in complex disorders like AD. Here, we have designed a multi-tier screening protocol combining ensemble docking to mine BACE1 inhibitor, as well as 2-D QSAR models for anti-amyloidogenic and antioxidant activities. An in house developed phytochemical library of 200 phytochemicals has been screened through this multi-target procedure which mine hesperidin, a flavanone glycoside commonly found in citrus food items, as a multi-potent phytochemical in AD therapeutics. Steady-state and time-resolved fluorescence spectroscopy reveal that binding of hesperidin to the active site of BACE1 induces a conformational transition of the protein from open to closed form. Hesperidin docks close to the catalytic aspartate residues and orients itself in a way that blocks the cavity opening thereby precluding substrate binding. Hesperidin is a high affinity BACE1 inhibitor and only 500 nM of the compound shows complete inhibition of the enzyme activity. Furthermore, ANS and Thioflavin-T binding assay show that hesperidin completely inhibits the amyloid fibril formation which is further supported by atomic force microscopy. Hesperidin exhibits moderate ABTS(+) radical scavenging assay but strong hydroxyl radical scavenging ability, as evident from DNA nicking assay. Present study demonstrates the applicability of a novel multi-target screening procedure to mine multi-potent agents from natural origin for AD therapeutics.

A role for peroxidasin PXN-1 in aspects of C. elegans development.

The Caenorhabditis elegans peroxidasins, PXN-1 and PXN-2, are extracellular peroxidases; pxn-2 is involved in muscle-epidermal attachment during embryonic morphogenesis and in specific axon guidance. Here we investigate potential roles of the other homologue of peroxidasin, pxn-1, in C. elegans. A pxn-1 deletion mutant showed high lethality under heat-stress conditions. Using a transcriptional GFP reporter, pxn-1 expression was observed in various tissues including neurons, muscles, and hypodermis. A translational fusion showed that PXN-1::GFP was secreted and localized in extracellular matrix, particularly along body wall muscles and pharyngeal muscles. Various neuronal developmental defects were observed in pxn-1 mutants and in pxn-1 over-expressing animals, including handedness, branching, breakage, tangling, and defasciculation. These results suggest that pxn-1, like other peroxidasins, plays an important role throughout development.

Nucleolar GTPase NOG-1 regulates development, fat storage, and longevity through insulin/IGF signaling in C. elegans.

NOG1 is a nucleolar GTPase that is critical for 60S ribosome biogenesis. Recently, NOG1 was identified as one of the downstream regulators of target of rapamycin (TOR) in yeast. It is reported that TOR is involved in regulating lifespan and fat storage in Caenorhabditis elegans. Here, we show that the nog1 ortholog (T07A9.9: nog-1) in C. elegans regulates growth, development, lifespan, and fat metabolism. A green fluorescence protein (GFP) promoter assay revealed ubiquitous expression of C. elegans nog-1 from the early embryonic to the adult stage. Furthermore, the GFP-tagged NOG-1 protein is localized to the nucleus, whereas the aberrant NOG-1 protein is concentrated in the nucleolus. Functional studies of NOG-1 in C. elegans further revealed that nog-1 knockdown resulted in smaller broodsize, slower growth, increased life span, and more fat storage. Moreover, nog-1 over-expression resulted in decreased life span. Taken together, our data suggest that nog-1 in C. elegans may be an important player in regulating life span and fat storage via the insulin/IGF pathway.

The multiple faces of calcineurin signaling in Caenorhabditis elegans: development, behaviour and aging.

Calcineurin, a well-conserved protein phosphatase 2B (PP2B), is a Ca2+-calmodulin-dependent serine/threonine protein phosphatase that is known to be involved in a myriad of cellular processes and signal transduction pathways. The biological role of calcineurin has been extensively studied in diverse groups of organisms. Homologues of mammalian and Drosophila calcineurin subunits exist in the nematode, Caenorhabditis elegans. The C. elegans counterpart of the catalytic subunit, calcineurin A, cna-1/tax-6, and the regulatory subunit, calcineurin B, cnb-1, are known to express ubiquitously in multiple tissues including neurons. The characterization of C. elegans calcineurin mutants facilitates identification of its physiological functions and signaling pathways. Genetic interactions between cna-1/tax-6 and cnb-1 mutants with a number of mutants involved in several signaling pathways have exemplified the pivotal role of calcineurin in regulating nematode development, behaviour and lifespan (aging). The present review has been aimed to provide a succinct summary of the multiple functions of calcineurin in C. elegans relating to its development, fertility, proliferation, behaviour and lifespan. Analyses of cna-1/tax-6 and cnb-1 interacting proteins and regulators of the phosphatase in this fascinating worm model have an immense scope to identify potential drug targets in various parasitic nematodes, which cause many diseases inflicting huge economic loss; and also for many human diseases, particularly neurodegenerative and myocardial diseases.

Two thioredoxin reductases, trxr-1 and trxr-2, have differential physiological roles in Caenorhabditis elegans.

Thioredoxin reductase (TrxR) is a member of the pyridine nucleotide-disulfide reductase family, which mainly functions in the thioredoxin system. TrxR is found in all living organisms and exists in two major ubiquitous isoenzymes in higher eukaryotic cells; One is cytosolic and the other mitochondrial. Mitochondrial TrxR functions to protect mitochondria from oxidative stress, where reactive oxidative species are mainly generated, while cytosolic TrxR plays a role to maintain optimal oxido-reductive status in cytosol. In this study, we report differential physiological functions of these two TrxRs in C. elegans. trxr-1, the cytosolic TrxR, is highly expressed in pharynx, vulva and intestine, whereas trxr-2, the mitochondrial TrxR, is mainly expressed in pharyngeal and body wall muscles. Deficiency of the non-selenoprotein trxr-2 caused defects in longevity and delayed development under stress conditions, while deletion mutation of the selenoprotein trxr-1 resulted in interference in acidification of lysosomal compartment in intestine. Interestingly, the acidification defect of trxr-1(jh143) deletion mutant was rescued, not only by selenocystein-containing wild type TRXR-1, but also cysteine-substituted mutant TRXR-1. Both trxr-1 and trxr-2 were up-regulated when worms were challenged by environmental stress such as heat shock. These results suggest that trxr-1 and trxr-2 function differently at organismal level presumably by their differential sub-cellular localization in C. elegans.

Functional assessment of Nramp-like metal transporters and manganese in Caenorhabditis elegans.

Nramp1 (natural resistance-associated macrophage protein-1) is a functionally conserved iron-manganese transporter in macrophages. Manganese (Mn), a superoxide scavenger, is required in trace amounts and functions as a cofactor for most antioxidants. Three Nramp homologs, smf-1, smf-2, and smf-3, have been identified thus far in the nematode Caenorhabditis elegans. A GFP promoter assay revealed largely intestinal expression of the smf genes from early embryonic through adult stages. In addition, smf deletion mutants showed increased sensitivity to excess Mn and mild sensitivity to EDTA. Interestingly, these smf deletion mutants demonstrated hypersensitivity to the pathogen Staphylococcus aureus, an effect that was rescued by Mn feeding or knockdown of the Golgi calcium/manganese ATPase, pmr-1, indicating that Mn uptake is essential for the innate immune system. This reversal of pathogen sensitivity by Mn feeding suggests a protective and therapeutic role of Mn in pathogen evasion systems. We propose that the C. elegans intestinal lumen may mimic the mammalian macrophage phagosome and thus could be a simple model for studying Mn-mediated innate immunity.

Opposing functions of calcineurin and CaMKII regulate G-protein signaling in egg-laying behavior of C.elegans.

Ca(2+)/calmodulin-dependent calcineurin has been shown to have important roles in various Ca(2+) signaling pathways. We have previously reported that cnb-1(jh103) mutants, null mutants of a regulatory B subunit, displayed pleiotropic defects including uncoordinated movement and delayed egg laying in Caenorhabditis elegans. Interestingly, gain-of-function mutants of a catalytic A subunit showed exactly opposite phenotypes to those of cnb-1(null) mutants providing an excellent genetic model to define calcium-mediated signaling pathway at the organism level. Furthermore, calcineurin is also important for normal cuticle formation, which is required for maintenance of normal body size in C.elegans. Genetic interactions between tax-6 and several mutants including egl-30 and egl-10, which are known to be involved in G-protein signaling pathways suggest that calcineurin indeed regulates locomotion and serotonin-mediated egg laying through goa-1(Goalpha) and egl-30(Gqalpha). Our results indicate that, along with CaMKII, calcineurin regulates G-protein-coupled phosphorylation signaling pathways in C.elegans.

Regulation of calcineurin, a calcium/calmodulin-dependent protein phosphatase, in C. elegans.

Calcineurin is a calcium/calmodulin-dependent serine/ threonine protein phosphatase. It is a heterodimeric protein consisting of a catalytic subunit calcineurin A, and a regulatory calcium-binding subunit, calcineurin B. The primary sequence of both subunits and heterodimeric structure is highly conserved from yeast to mammals. Calcineurin has long been implicated in various signaling pathways. Calcineurin genes (cna-1/tax-6 and cnb-1) have been identified in the nematode Caenorhabditis elegans, which share high homology with their Drosophila and mammalian counterparts. C. elegans calcineurin binds calcium and functions as a heterodimeric protein phosphatase establishing its biochemical conservation. Calcineurin expresses in diverse tissues implicating its important role in various physiological processes. This review will focus in brief on the expression pattern and regulation of calcineurin including its effect on growth and development, locomotion, egg-laying, and sensory responses.

The Caenorhabditis elegans homologue of Down syndrome critical region 1, RCN-1, inhibits multiple functions of the phosphatase calcineurin.

A conserved family of calcineurin-regulating proteins whose members have been implicated in several disease models such as Down syndrome, Alzheimer's disease, and cardiac hypertrophy has been identified in several organisms including yeast, mice, and humans. We have characterized Caenorhabditis elegans rcn-1, which belongs to this family of calcineurin regulators, and shows approximately 40% identity with the human homologue DSCR-1. rcn-1 is expressed in hypodermal cells, nerve cords and various neurons, vulva epithelial and muscle cells, marginal cells of the pharynx, and structures of the male tail. rcn-1 expression is upregulated by calcineurin activity. RCN-1 binds to calcineurin A from C.elegans lysate in a calcium-dependent manner, and inhibits bovine calcineurin phosphatase activity dose-dependently. In addition, overexpression of RCN-1 results in calcineurin-deficient phenotypes such as small body size, cuticle defects, fertility defects, slow growth, and serotonin-resistant egg-laying defects. Moreover, phenotypes observed in gain-of-function calcineurin mutant animals were restored to normal by RCN-1 overexpression. These results demonstrate an effective and specific inhibition of calcineurin in vitro as well as in vivo by RCN-1.