Priti Talwar

A Molecular Web: Endoplasmic Reticulum Stress, Inflammation, and Oxidative Stress

Execution of fundamental cellular functions demands regulated protein folding homeostasis. Endoplasmic reticulum (ER) is an active organelle existing to implement this function by folding and modifying secretory and membrane proteins. Loss of protein folding homeostasis is central to various diseases and budding evidences suggest ER stress as being a major contributor in the development or pathology of a diseased state besides other cellular stresses. The trigger for diseases may be diverse but, inflammation and/or ER stress may be basic mechanisms increasing the severity or complicating the condition of the disease. Chronic ER stress and activation of the unfolded-protein response (UPR) through endogenous or exogenous insults may result in impaired calcium and redox homeostasis, oxidative stress via protein overload thereby also influencing vital mitochondrial functions. Calcium released from the ER augments the production of mitochondrial Reactive Oxygen Species (ROS). Toxic accumulation of ROS within ER and mitochondria disturbs fundamental organelle functions. Sustained ER stress is known to potentially elicit inflammatory responses via UPR pathways. Additionally, ROS generated through inflammation or mitochondrial dysfunction could accelerate ER malfunction. Dysfunctional UPR pathways have been associated with a wide range of diseases including several neurodegenerative diseases, stroke, metabolic disorders, cancer, inflammatory disease, diabetes mellitus, cardiovascular disease, and others. In this review, we have discussed the UPR signaling pathways, and networking between ER stress-induced inflammatory pathways, oxidative stress, and mitochondrial signaling events, which further induce or exacerbate ER stress.

Death Associated Protein Kinase 1 (DAPK1): A Regulator of Apoptosis and Autophagy

Death-Associated Protein Kinase 1 (DAPK1) belongs to a family of five serine/threonine (Ser/Thr) kinases that possess tumor suppressive function and also mediate a wide range of cellular processes, including apoptosis and autophagy. The loss and gain-of–function of DAPK1 is associated with various cancer and neurodegenerative diseases respectively. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms involved in DAPK1-mediated autophagy/apoptosis. In the present review, we have discussed the structural information and various cellular functions of DAPK1 in a comprehensive manner.

Synthetic Triterpenoid Cyano Enone of Methyl Boswellate Activates Intrinsic, Extrinsic, and Endoplasmic Reticulum Stress Cell Death Pathways in Tumor Cell Lines

We explored the effect of a novel synthetic triterpenoid compound cyano enone of methyl boswellates (CEMB) on various prostate cancer and glioma cancer cell lines. CEMB displayed concentration-dependent cytotoxic activity with submicromolar lethal dose 50% (LD50) values in 10 of 10 tumor cell lines tested. CEMB-induced cytotoxicity is accompanied by activation of downstream effector caspases (caspases 3 and 7) and by upstream initiator caspases involved in both the extrinsic (caspase 8) and intrinsic (caspase 9) apoptotic pathways. By using short interfering RNAs (siRNA), we show evidence that knockdown of caspase 8, DR4, Apaf-1, and Bid impairs CEMB-induced cell death. Similar to other proapoptotic synthetic triterpenoid compounds, CEMB-induced apoptosis involved endoplasmic reticulum stress, as shown by partial rescue of tumor cells by siRNA-mediated knockdown of expression of genes involved in the unfolded protein response such as IRE1α, PERK, and ATF6. Altogether, our results suggest that CEMB stimulates several apoptotic pathways in cancer cells, suggesting that this compound should be evaluated further as a potential agent for cancer therapy. Mol Cancer Ther; 10(9); 1635–43. ©2011 AACR.

In silico identification of genetic variants in glucocerebrosidase (GBA) gene involved in Gaucher's disease using multiple software tools

Gauchers disease (GD) is an autosomal recessive disorder caused by the deficiency of glucocerebrosidase, a lysosomal enzyme that catalyses the hydrolysis of the glycolipid glucocerebroside to ceramide and glucose. Polymorphisms in GBA gene have been associated with the development of Gaucher disease. We hypothesize that prediction of SNPs using multiple state of the art software tools will help in increasing the confidence in identification of SNPs involved in GD. Enzyme replacement therapy is the only option for GD. Our goal is to use several state of art SNP algorithms to predict/address harmful SNPs using comparative studies. In this study seven different algorithms (SIFT, MutPred, nsSNP Analyzer, PANTHER, PMUT, PROVEAN, and SNPs&GO) were used to predict the harmful polymorphisms. Among the seven programs, SIFT found 47 nsSNPs as deleterious, MutPred found 46 nsSNPs as harmful. nsSNP Analyzer program found 43 out of 47 nsSNPs are disease causing SNPs whereas PANTHER found 32 out of 47 as highly deleterious, 22 out of 47 are classified as pathological mutations by PMUT, 44 out of 47 were predicted to be deleterious by PROVEAN server, all 47 shows the disease related mutations by SNPs&GO. Twenty two nsSNPs were commonly predicted by all the seven different algorithms. The common 22 targeted mutations are F251L, C342G, W312C, P415R, R463C, D127V, A309V, G46E, G202E, P391L, Y363C, Y205C, W378C, I402T, S366R, F397S, Y418C, P401L, G195E, W184R, R48W, and T43R.

Exploring putative inhibitors of Death Associated Protein Kinase 1 (DAPK1) via targeting Gly-Glu-Leu (GEL) and Pro-Glu-Asn (PEN) substrate recognition motifs

Recently, a new signaling complex Death Associated Protein Kinase 1 (DAPK1) ̶ N-methyl-D-aspartate receptor subtype 2B (NMDAR2B or NR2B) engaged in the neuronal death cascade was identified and it was found that after stroke injury, N-methyl-D-aspartate glutamate (NMDA) receptors interact with DAPK1 through NR2B subunit and lead to excitotoxicity via over-activation of NMDA receptors. An acute brain injury, such as stroke, is a serious life-threatening medical condition which occurs due to poor blood supply to the brain and further leads to neuronal cell death. During a stroke, activated DAPK1 migrates towards the extra-synaptic site and binds to NR2B subunit of NMDA receptor. It is this DAPK1-NR2B interaction that arbitrates the pathological processes like apoptosis, necrosis, and autophagy of neuronal cells observed in stroke injury, hence we aimed to inhibit this vital interaction to prevent neuronal damage. In the present study, using PubChem database, we applied an integrative approach of virtual screening and molecular dynamic simulations and identified a potential lead compound 11 that interrupts DAPK1-NR2B interaction by competing with both ATP and substrate for their binding sites on DAPK1. This inhibitor was found potent and considerably selective to DAPK1 as it made direct contact with the ATP binding sites as well as substrate recognition motifs: Gly-Glu-Leu (GEL) and Pro-Glu-Asn (PEN). Further in vitro and in vivo experiments are demanded to validate the efficacy of compound 11 nevertheless, it can be considered as suitable starting point for designing DAPK1 inhibitors.

IRE1α is critical for kaempferol induced neuroblastoma differentiation

Neuroblastoma is an embryonic malignancy arises out of the neural crest cells of the sympathetic nervous system. It is the most common childhood tumor and well known for its spontaneous regression via the process of differentiation. The induction of differentiation using small molecule modulators such as all trans retinoic acid is one of the treatment strategies to treat the residual disease. In this study, we have reported the effect of kaempferol, a phytoestrogen in inducing differentiation of neuroblastoma cells in vitro. Treatment of neuroblastoma cells with kaempferol reduced the proliferation and enhanced apoptosis along with the induction of neuritogenesis. Analysis of the expression of neuron specific markers such as β III tubulin, neuron specific enolase and NRDG1 (N-myc down regulated gene 1) revealed the process of differentiation accompanying kaempferol induced apoptosis. Further analysis on understanding the molecular mechanism of action showed that the activity of kaempferol happened through the activation of the endoribonuclease activity of IRE1α (Inositol requiring enzyme 1 alpha), an endoplasmic reticulum (ER) resident transmembrane protein. The in silico docking analysis and biochemical assays using recombinant human IRE1α confirms the binding of kaempferol to the ATP binding site of IRE1α and thereby activating ribonuclease activity. Treatment of cells with the small molecule inhibitor STF083010 which specifically targets and inhibits the endoribonuclease activity of IRE1α showed reduced expression of neuron specific markers and curtailed neuritogenesis. The knock down of IRE1α using plasmid based shRNA lentiviral particles also showed diminished changes in the change in morphology of the cells upon kaempferol treatment. Thus our study suggests that kaempferol induces differentiation of neuroblastoma cells via the IRE1α-XBP1 pathway.

Identification of FDA-approved drugs as novel allosteric inhibitors of human executioner caspases

The regulation of apoptosis is a tightly coordinated process and caspases are its chief regulators. Of special importance are the executioner caspases, caspase‐3/7, the activation of which irreversibly sets the cell on the path of death. Dysregulation of apoptosis, particularly an increased rate of cell death lies at the root of numerous human diseases. Although several peptide‐based inhibitors targeting the homologous active site region of caspases have been developed, owing to their non‐specific activity and poor pharmacological properties their use has largely been restricted. Thus, we sought to identify FDA‐approved drugs that could be repurposed as novel allosteric inhibitors of caspase‐3/7. In this study, we virtually screened a catalog of FDA‐approved drugs targeting an allosteric pocket located at the dimerization interface of caspase‐3/7. From among the top‐scoring hits we short‐listed 5 compounds for experimental validation. Our enzymatic assays using recombinant caspase‐3 suggested that 4 out of the 5 drugs effectively inhibited caspase‐3 enzymatic activity in vitro with IC50 values ranging ~10‐55 μM. Structural analysis of the docking poses show the 4 compounds forming specific non‐covalent interactions at the allosteric pocket suggesting that these molecules could disrupt the adjacently‐located active site. In summary, we report the identification of 4 novel non‐peptide allosteric inhibitors of caspase‐3/7 from among FDA‐approved drugs.

Identification of ASB7 as ER stress responsive gene through a genome wide in silico screening for genes with ERSE

The endoplasmic reticulum (ER) not only performs its basic function of regulating calcium homeostasis, lipid biosynthesis, folding, modifying and transporting proteins but also plays a decisive role in regulating multiple cellular processes ranging from cell growth and differentiation to apoptosis and autophagy. Disturbances in ER homeostasis initiate the unfolded protein response (UPR) implicated in the pathogenesis of many human diseases. Drugging the UPR components for therapeutic interventions has received considerable attention. The purpose of this study is to identify genes that are previously unsuspected to be regulated under ER stress. Because ER stress-inducible gene expression is majorly regulated under ERSE elements, we screened human genome by adopting an in silico approach using ERSE elements (I, II, III) as probes and identified 337 candidate genes. Having knowledge of the importance of E3 ubiquitin ligase in the ERAD machinery; we validated our preliminary search by focusing on one of the hits i.e. ASB7 gene that encodes E3 ubiquitin ligase. In HeLa cells, we found that pharmacological induction of ER stress led to an increase in the expression of ASB7 with simultaneous activation of UPR pathways. Although knockdown of ASB7 expression leads to significant reduction in GRP78 and CHOP mRNA levels, it did not protect cells from ER stress-induced cell death. Also, an up-regulation in the expression of pro-inflammatory genes like TNF-α and IL-1β in ASB7 knockdown cells was observed under ER stress. Collectively, our findings suggest that ASB7 is regulated under ER stress and this study also identifies several other genes that could apparently be regulated under ER stress.

Multifaceted roles of ASB proteins and its pathological significance

BackgroundPost-translational (PT) modification in cells regulates many intracellular events like signal transduction, transcription, cell cycle, protein quality control, apoptosis and cellular development. Ubiquitination is one of the PT modifications which functions as a marker for degradation of target proteins by the proteasome and as a regulatory mechanism for several signalling pathways. The ubiquitination mechanism requires multiple enzymes, including E1, E2, and E3 ligases. Among them, E3 ligases play a major role in recognizing target proteins and an essential feature of protein homeostatic mechanisms within the cell. Most of the ASB (ankyrin repeat SOCS box) proteins function as RING family of E3 ubiquitin ligases characterized by the presence of two conserved domains N-terminal ankyrin repeat and C-terminal SOCS box domainMethods and ResultsCurrent studies have shown that some ASBs function as important regulators of several signalling pathways. This review gives an overview of ASB proteins on numerous cellular processes such as insulin signalling, spermatogenesis, myogenesis and in cellular development. Including various pathological situations, such as cancer, primary open-angle glaucoma, and inflammation, indicating that ASBs has important functions in both normal and pathological developmentConclusionsThis article provides a precise comprehensive focus on ASBs protein structure, its biological functions, and their pathological significance.

CDDO and ATRA Instigate Differentiation of IMR32 Human Neuroblastoma Cells

Neuroblastoma is the most common solid extra cranial tumor in infants. Improving the clinical outcome of children with aggressive tumors undergoing one of the multiple treatment options has been a major concern. Differentiating neuroblastoma cells holds promise in inducing tumor growth arrest and treating minimal residual disease. In this study, we investigated the effect of partial PPARγ agonist 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) on human neuroblastoma IMR32 cells. Our results demonstrate that treatment with low concentration of CDDO and particularly in combination with all trans retinoic acid (ATRA) induced neurite outgrowth, increased the percentage of more than two neurites bearing cells, and decreased viability in IMR32 cells. These morphological changes were associated with an increase in expression of bonafide differentiation markers like β3-tubulin and Neuron Specific Enolase (NSE). The differentiation was accompanied by a decrease in the expression of MYCN whose amplification is known to contribute to the pathogenesis of neuroblastoma. MYCN is known to negatively regulate NMYC downstream-regulated gene 1 (NDRG1) in neuroblastomas. MYCN down-regulation induced by CDDO correlated with increased expression of NDRG1. CDDO decreased Anaplastic Lymphoma Kinase (ALK) mRNA expression without affecting its protein level, while ATRA significantly down-regulated ALK. Antagonism of PPARγ receptor by T0070907 meddled with differentiation inducing effects of CDDO as observed by stunted neurite growth, increased viability and decreased expression of differentiation markers. Our findings indicate that IMR32 differentiation induced by CDDO in combination with ATRA enhances, differentiation followed by cell death via cAMP-response-element binding protein (CREB) independent and PPARγ dependent signaling mechanisms.