G., Krishna, K., Gupta, N., Soong, T. W., Mallilankaraman, K., Sajikumar, S., & Dheen, S. T. (2019). Epigenetic regulation of microglial phosphatidylinositol 3‐kinase pathway involved in long‐term potentiation and synaptic plasticity in rats.

Abstract

Microglia are the main form of immune defense in the central nervous system. Microglia express phosphatidylinositol 3-kinase (PI3K), which has been shown to play a significant role in synaptic plasticity in neurons and inflammation via microglia. This study shows that microglial PI3K is regulated epigenetically through histone modifications and posttranslationally through sumoylation and is involved in long-term potentiation (LTP) by modulating the expression of brain-derived neurotrophic factor (BDNF), which has been shown to be involved in neuronal synaptic plasticity. Sodium butyrate, a histone deacetylase inhibitor, upregulates PI3K expression, the phosphorylation of its downstream effectors, AKT and cAMP response element-binding protein (CREB), and the expression of BDNF in microglia, suggesting that BDNF secretion is regulated in microglia via epigenetic regulation of PI3K. Further, knockdown of SUMO1 in BV2 microglia results in a decrease in the expression of PI3K, the phosphorylation of AKT and CREB, as well as the expression of BDNF. These results suggest that microglial PI3K is epigenetically regulated by histone modifications and posttranslationally modified by sumoylation, leading to altered expression of BDNF. Whole-cell voltage-clamp showed the involvement of microglia in neuronal LTP, as selective ablation or disruption of microglia with clodronate in rat hippocampal slices abolished LTP. However, LTP was rescued when the same hippocampal slices were treated with active PI3K or BDNF, indicating that microglial PI3K/AKT signaling contributes to LTP and synaptic plasticity. Understanding the mechanisms by which microglial PI3K influences synapses provides insights into the ways it can modulate synaptic transmission and plasticity in learning and memory.

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Kandilya, D., Maskomani, S., Shyamasundar, S., Tambyah, P. A., Yng, C. S., Lee, R. C. H., … Dheen, S. T. (2019). Zika virus alters DNA methylation status of genes involved in Hippo signaling pathway in human neural progenitor cells.

Abstract

Aim: This study was aimed to understand if Zika virus (ZIKV) alters the DNA methylome of human neural progenitor cells (hNPCs). Materials & methods: Whole genome DNA methylation profiling was performed using human methylationEPIC array in control and ZIKV infected hNPCs. Results & conclusion: ZIKV infection altered the DNA methylation of several genes such as WWTR1 (TAZ) and RASSF1 of Hippo signaling pathway which regulates organ size during brain development, and decreased the expression of several centrosomal-related microcephaly genes, and genes involved in stemness and differentiation in human neural progenitor cells. Overall, ZIKV downregulated the Hippo signaling pathway genes which perturb the stemness and differentiation process in hNPCs, which could form the basis for ZIKV-induced microcephaly.

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Zeng, Q., Mallilankaraman, K., & Schwarz, H. (2019). Increased Akt-Driven Glycolysis Is the Basis for the Higher Potency of CD137L-DCs.

Abstract

CD137 ligand-induced dendritic cells (CD137L-DCs) are a new type of dendritic cells (DCs) that induce strong cytotoxic T cell responses. Investigating the metabolic activity as a potential contributing factor for their potency, we find a significantly higher rate of glycolysis in CD137L-DCs than in granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 induced monocyte-derived DCs (moDCs). Using unbiased screening, Akt-mTORC1 activity was found to be significantly higher throughout the differentiation and maturation of CD137L-DCs than that of moDCs. Furthermore, this higher activity of the Akt-mTORC1 pathway is responsible for the significantly higher glycolysis rate in CD137L-DCs than in moDCs. Inhibition of Akt during maturation or inhibition of glycolysis during and after maturation resulted in suppression of inflammatory DCs, with mature CD137L-DCs being the most affected ones. mTORC1, instead, was indispensable for the differentiation of both CD137L-DCs and moDCs. In contrast to its role in supporting lipid synthesis in murine bone marrow-derived DCs (BMDCs), the higher glycolysis rate in CD137L-DCs does not lead to a higher lipid content but rather to an accumulation of succinate and serine. These data demonstrate that the increased Akt-driven glycolysis underlies the higher activity of CD137L-DCs.

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Balaganapathy, P., Baik, S., Mallilankaraman, K., Sobey, C., Jo, D. and Arumugam, T. (2017). Interplay between Notch and p53 promotes neuronal cell death in ischemic stroke. Journal of Cerebral Blood Flow & Metabolism

Abstract

Stroke is the world's second leading cause of mortality, with a high incidence of morbidity. Numerous neuronal membrane receptors are activated by endogenous ligands and may contribute to infarct development. Notch is a well-characterized membrane receptor involved in cell differentiation and proliferation, and now shown to play a pivotal role in cell death during ischemic stroke. Blockade of Notch signaling by inhibition of γ-secretase, an enzyme that generates the active form of Notch, is neuroprotective following stroke. We have also identified that Pin1, a peptidyl-prolyl isomerase that regulates p53 transactivation under stress, promotes the pathogenesis of ischemic stroke via Notch signaling. Moreover, Notch can also mediate cell death through a p53-dependent pathway, resulting in apoptosis of neural progenitor cells. The current study has investigated the interplay between Notch and p53 under ischemic stroke conditions. Using pharmacological inhibitors, we have demonstrated that a Notch intracellular domain (NICD)/p53 interaction is involved in transcriptional regulation of genes downstream of p53 and NICD to modify stroke severity. Furthermore, the NICD/p53 interaction confers stability to p53 by rescuing it from ubiquitination. Together, these results indicate that Notch contributes to the pathogenesis of ischemic stroke by promoting p53 stability and signaling.

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Vais, H., Mallilankaraman, K., Mak, D., Hoff, H., Payne, R., Tanis, J. and Foskett, J. (2016). EMRE Is a Matrix Ca 2+ Sensor that Governs Gatekeeping of the Mitochondrial Ca 2+ Uniporter. Cell Reports, 14(3), pp.403-410.

Abstract

The mitochondrial uniporter (MCU) is an ion channel that mediates Ca(2+) uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca(2+) signaling. Matrix Ca(2+) concentration is similar to that in cytoplasm, despite an enormous driving force for entry, but the mechanisms that prevent mitochondrial Ca(2+) overload are unclear. Here, we show that MCU channel activity is governed by matrix Ca(2+) concentration through EMRE. Deletion or charge neutralization of its matrix-localized acidic C terminus abolishes matrix Ca(2+) inhibition of MCU Ca(2+) currents, resulting in MCU channel activation, enhanced mitochondrial Ca(2+) uptake, and constitutively elevated matrix Ca(2+) concentration. EMRE-dependent regulation of MCU channel activity requires intermembrane space-localized MICU1, MICU2, and cytoplasmic Ca(2+). Thus, mitochondria are protected from Ca(2+) depletion and Ca(2+) overload by a unique molecular complex that involves Ca(2+) sensors on both sides of the inner mitochondrial membrane, coupled through EMRE.

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Mallilankaraman, K., Cárdenas, C., Doonan, P., Chandramoorthy, H., Irrinki, K., Golenár, T., Csordás, G., Madireddi, P., Yang, J., Müller, M., Miller, R., Kolesar, J., Molgó, J., Kaufman, B., Hajnóczky, G., Foskett, J. and Madesh, M. (2015). Corrigenda: MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism. Nature Cell Biology, 17(7), pp.953-953.

Abstract

Ca(2+) flux across the mitochondrial inner membrane regulates bioenergetics, cytoplasmic Ca(2+) signals and activation of cell death pathways. Mitochondrial Ca(2+) uptake occurs at regions of close apposition with intracellular Ca(2+) release sites, driven by the inner membrane voltage generated by oxidative phosphorylation and mediated by a Ca(2+) selective ion channel (MiCa; ref. ) called the uniporter whose complete molecular identity remains unknown. Mitochondrial calcium uniporter (MCU) was recently identified as the likely ion-conducting pore. In addition, MICU1 was identified as a mitochondrial regulator of uniporter-mediated Ca(2+) uptake in HeLa cells. Here we identified CCDC90A, hereafter referred to as MCUR1 (mitochondrial calcium uniporter regulator 1), an integral membrane protein required for MCU-dependent mitochondrial Ca(2+) uptake. MCUR1 binds to MCU and regulates ruthenium-red-sensitive MCU-dependent Ca(2+) uptake. MCUR1 knockdown does not alter MCU localization, but abrogates Ca(2+) uptake by energized mitochondria in intact and permeabilized cells. Ablation of MCUR1 disrupts oxidative phosphorylation, lowers cellular ATP and activates AMP kinase-dependent pro-survival autophagy. Thus, MCUR1 is a critical component of a mitochondrial uniporter channel complex required for mitochondrial Ca(2+) uptake and maintenance of normal cellular bioenergetics.

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Vijayachari, P., Vedhagiri, K., Mallilankaraman, K., Mathur, P., Sardesai, N., Weiner, D., Ugen, K. and Muthumani, K. (2015). Immunogenicity of a novel enhanced consensus DNA vaccine encoding the leptospiral protein LipL45. Human Vaccines & Immunotherapeutics, 11(8), pp.1945-1953.

Abstract

Leptospirosis is a bacterial zoonotic disease caused by an infection with a spirochete belonging to the genus Leptospira. In animals, leptospirosis displays a wide range of pathologies, including fever, abortion, icterus, and uveitis. Conversely, infection in humans is associated with multi-organ injury, resulting in an increased rate of fatalities. Pathogenic leptospires are able to translocate through cell monolayers at a rate significantly greater than that of non-pathogenic leptospires. Thus, vaccine approaches have been focused on targeting bacterial motility, lipopolysaccharides (LPSs), lipoproteins, outer-membrane proteins (OMPs) and other potential virulence factors. Previous studies have indicated that leptospiral proteins elicit long-lasting immunological memory in infected humans. In the study reported here, the efficacy of a synthetic consensus DNA vaccine developed against the Leptospira membrane lipoprotein LipL45 was tested. After in vivo electroporation (EP) mediated intramuscular immunization with a synthetic LipL45 DNA vaccine (pLipL45) immunized mice developed a significant cellular response along with the development of anti-LipL45-specific antibodies. Specifically, the pLipL45 vaccine induced a significant Th1 type immune response, indicated by the higher production of IL-12 and IFN-γ cytokines. The results presented here are the first demonstration that a LipL45 based DNA immunogen has potential as a anti-Leptospira vaccine.

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Cheng, Z., Jiang, X., Pansuria, M., Fang, P., Mai, J., Mallilankaraman, K., Gandhirajan, R., Eguchi, S., Scalia, R., Madesh, M., Yang, X. and Wang, H. (2014). Hyperhomocysteinemia and Hyperglycemia Induce and Potentiate Endothelial Dysfunction via μ-Calpain Activation. Diabetes, 64(3), pp.947-959.

Abstract

Plasma homocysteine (Hcy) levels are positively correlated with cardiovascular mortality in diabetes. However, the joint effect of hyperhomocysteinemia (HHcy) and hyperglycemia (HG) on endothelial dysfunction (ED) and the underlying mechanisms have not been studied. Mild (22 µmol/L) and moderate (88 µmol/L) HHcy were induced in cystathionine β-synthase wild-type (Cbs(+/+)) and heterozygous-deficient (Cbs(-/+)) mice by a high-methionine (HM) diet. HG was induced by consecutive injection of streptozotocin. We found that HG worsened HHcy and elevated Hcy levels to 53 and 173 µmol/L in Cbs(+/+) and Cbs(-/+) mice fed an HM diet, respectively. Both mild and moderate HHcy aggravated HG-impaired endothelium-dependent vascular relaxation to acetylcholine, which was completely abolished by endothelial nitric oxide synthase (eNOS) inhibitor N(G)-nitro-L-arginine methyl ester. HHcy potentiated HG-induced calpain activation in aortic endothelial cells isolated from Cbs mice. Calpain inhibitors rescued HHcy- and HHcy/HG-induced ED in vivo and ex vivo. Moderate HHcy- and HG-induced μ-calpain activation was potentiated by a combination of HHcy and HG in the mouse aorta. μ-Calpain small interfering RNA (μ-calpsiRNA) prevented HHcy/HG-induced ED in the mouse aorta and calpain activation in human aortic endothelial cells (HAECs) treated with DL-Hcy (500 µmol/L) and d-glucose (25 mmol) for 48 h. In addition, HHcy accelerated HG-induced superoxide production as determined by dihydroethidium and 3-nitrotyrosin staining and urinary 8-isoprostane/creatinine assay. Antioxidants rescued HHcy/HG-induced ED in mouse aortas and calpain activation in cultured HAECs. Finally, HHcy potentiated HG-suppressed nitric oxide production and eNOS activity in HAECs, which were prevented by calpain inhibitors or μ-calpsiRNA. HHcy aggravated HG-increased phosphorylation of eNOS at threonine 497/495 (eNOS-pThr497/495) in the mouse aorta and HAECs. HHcy/HG-induced eNOS-pThr497/495 was reversed by µ-calpsiRNA and adenoviral transduced dominant negative protein kinase C (PKC)β2 in HAECs. HHcy and HG induced ED, which was potentiated by the combination of HHcy and HG via μ-calpain/PKCβ2 activation-induced eNOS-pThr497/495 and eNOS inactivation.

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Miller, B., Hoffman, N., Merali, S., Zhang, X., Wang, J., Rajan, S., Shanmughapriya, S., Gao, E., Barrero, C., Mallilankaraman, K., Song, J., Gu, T., Hirschler-Laszkiewicz, I., Koch, W., Feldman, A., Madesh, M. and Cheung, J. (2014). TRPM2 Channels Protect against Cardiac Ischemia-Reperfusion Injury. Journal of Biological Chemistry, 289(11), pp.7615-7629.

Abstract

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca(2+) uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca(2+) uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca(2+) uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels.

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Hoffman, N., Chandramoorthy, H., Shamugapriya, S., Zhang, X., Rajan, S., Mallilankaraman, K., Gandhirajan, R., Vagnozzi, R., Ferrer, L., Sreekrishnanilayam, K., Natarajaseenivasan, K., Vallem, S., Force, T., Choi, E., Cheung, J. and Madesh, M. (2013). MICU1 Motifs Define Mitochondrial Calcium Uniporter Binding and Activity. Cell Reports, 5(6), pp.1576-1588.

Abstract

Resting mitochondrial matrix Ca(2+) is maintained through a mitochondrial calcium uptake 1 (MICU1)-established threshold inhibition of mitochondrial calcium uniporter (MCU) activity. It is not known how MICU1 interacts with MCU to establish this Ca(2+) threshold for mitochondrial Ca(2+) uptake and MCU activity. Here, we show that MICU1 localizes to the mitochondrial matrix side of the inner mitochondrial membrane and MICU1/MCU binding is determined by a MICU1 N-terminal polybasic domain and two interacting coiled-coil domains of MCU. Further investigation reveals that MICU1 forms homo-oligomers, and this oligomerization is independent of the polybasic region. However, the polybasic region confers MICU1 oligomeric binding to MCU and controls mitochondrial Ca(2+) current (IMCU). Moreover, MICU1 EF hands regulate MCU channel activity, but do not determine MCU binding. Loss of MICU1 promotes MCU activation leading to oxidative burden and a halt to cell migration. These studies establish a molecular mechanism for MICU1 control of MCU-mediated mitochondrial Ca(2+) accumulation, and dysregulation of this mechanism probably enhances vascular dysfunction.

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Vagnozzi, R., Gatto, G., Kallander, L., Hoffman, N., Mallilankaraman, K., Ballard, V., Lawhorn, B., Stoy, P., Philp, J., Graves, A., Naito, Y., Lepore, J., Gao, E., Madesh, M. and Force, T. (2013). Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K Limits Oxidative Stress, Injury, and Adverse Remodeling in the Ischemic Heart. Science Translational Medicine, 5(207)

Abstract

Percutaneous coronary intervention is first-line therapy for acute coronary syndromes (ACS) but can promote cardiomyocyte death and cardiac dysfunction via reperfusion injury, a phenomenon driven in large part by oxidative stress. Therapies to limit this progression have proven elusive, with no major classes of new agents since the development of anti-platelets/anti-thrombotics. We report that cardiac troponin I-interacting kinase (TNNI3K), a cardiomyocyte-specific kinase, promotes ischemia/reperfusion injury, oxidative stress, and myocyte death. TNNI3K-mediated injury occurs through increased mitochondrial superoxide production and impaired mitochondrial function and is largely dependent on p38 mitogen-activated protein kinase (MAPK) activation. We developed a series of small-molecule TNNI3K inhibitors that reduce mitochondrial-derived superoxide generation, p38 activation, and infarct size when delivered at reperfusion to mimic clinical intervention. TNNI3K inhibition also preserves cardiac function and limits chronic adverse remodeling. Our findings demonstrate that TNNI3K modulates reperfusion injury in the ischemic heart and is a tractable therapeutic target for ACS. Pharmacologic TNNI3K inhibition would be cardiac-selective, preventing potential adverse effects of systemic kinase inhibition.

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Barrero, C., Perez-Leal, O., Aksoy, M., Moncada, C., Ji, R., Lopez, Y., Mallilankaraman, K., Madesh, M., Criner, G., Kelsen, S. and Merali, S. (2013). Histone 3.3 Participates in a Self-Sustaining Cascade of Apoptosis That Contributes to the Progression of Chronic Obstructive Pulmonary Disease. American Journal of Respiratory and Critical Care Medicine, 188(6), pp.673-683.

Abstract

RATIONALE: Shifts in the gene expression of nuclear protein in chronic obstructive pulmonary disease (COPD), a progressive disease that is characterized by extensive lung inflammation and apoptosis, are common; however, the extent of the elevation of the core histones, which are the major components of nuclear proteins and their consequences in COPD, has not been characterized, which is important because extracellular histones are cytotoxic to endothelial and airway epithelial cells.

OBJECTIVES: To investigate the role of extracellular histones in COPD disease progression.

METHODS: We analyzed the nuclear lung proteomes of ex-smokers with and without the disease. Further studies on the consequences of H3.3 were also performed.

MEASUREMENTS AND MAIN RESULTS: A striking finding was a COPD-specific eightfold increase of hyperacetylated histone H3.3. The hyperacetylation renders H3.3 resistant to proteasomal degradation despite ubiquitination; when combined with the reduction in proteasome activity that is known for COPD, this resistance helps account for the increased levels of H3.3. Using anti-H3 antibodies, we found H3.3 in the airway lumen, alveolar fluid, and plasma of COPD samples. H3.3 was cytotoxic to lung structural cells via a mechanism that involves the perturbation of Ca(2+) homeostasis and mitochondrial toxicity. We used the primary human airway epithelial cells and found that the antibodies to either the C or N terminus of H3 could partially reverse H3.3 toxicity.

CONCLUSIONS: Our data indicate that there is an uncontrolled positive feedback loop in which the damaged cells release acetylated H3.3, which causes more damage, adds H3.3 release, and contributes toward the disease progression.

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Miller, B., Wang, J., Hirschler-Laszkiewicz, I., Gao, E., Song, J., Zhang, X., Koch, W., Madesh, M., Mallilankaraman, K., Gu, T., Chen, S., Keefer, K., Conrad, K., Feldman, A. and Cheung, J. (2013). The second member of transient receptor potential-melastatin channel family protects hearts from ischemia-reperfusion injury. AJP: Heart and Circulatory Physiology, 304(7), pp.H1010-H1022.

Abstract

The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca(2+) influx that was dependent on extracellular Ca(2+), was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/dt between WT and KO hearts. At 2-3 days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/dt compared with WT hearts. Compared with WT I/R myocytes, expression of the Na(+)/Ca(2+) exchanger (NCX1) and NCX1 current were increased, expression of the α1-subunit of Na(+)-K(+)-ATPase and Na(+) pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca(2+) concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (SODs) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/R-induced oxidative stress.

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Gandhirajan, R., Meng, S., Chandramoorthy, H., Mallilankaraman, K., Mancarella, S., Gao, H., Razmpour, R., Yang, X., Houser, S., Chen, J., Koch, W., Wang, H., Soboloff, J., Gill, D. and Madesh, M. (2013). Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation. Journal of Clinical Investigation.

Abstract

During sepsis, acute lung injury (ALI) results from activation of innate immune cells and endothelial cells by endotoxins, leading to systemic inflammation through proinflammatory cytokine overproduction, oxidative stress, and intracellular Ca2+ overload. Despite considerable investigation, the underlying molecular mechanism(s) leading to LPS-induced ALI remain elusive. To determine whether stromal interaction molecule 1-dependent (STIM1-dependent) signaling drives endothelial dysfunction in response to LPS, we investigated oxidative and STIM1 signaling of EC-specific Stim1-knockout mice. Here we report that LPS-mediated Ca2+ oscillations are ablated in ECs deficient in Nox2, Stim1, and type II inositol triphosphate receptor (Itpr2). LPS-induced nuclear factor of activated T cells (NFAT) nuclear accumulation was abrogated by either antioxidant supplementation or Ca2+ chelation. Moreover, ECs lacking either Nox2 or Stim1 failed to trigger store-operated Ca2+ entry (SOCe) and NFAT nuclear accumulation. LPS-induced vascular permeability changes were reduced in EC-specific Stim1-/- mice, despite elevation of systemic cytokine levels. Additionally, inhibition of STIM1 signaling prevented receptor-interacting protein 3-dependent (RIP3-dependent) EC death. Remarkably, BTP2, a small-molecule calcium release-activated calcium (CRAC) channel blocker administered after insult, halted LPS-induced vascular leakage and pulmonary edema. These results indicate that ROS-driven Ca2+ signaling promotes vascular barrier dysfunction and that the SOCe machinery may provide crucial therapeutic targets to limit sepsis-induced ALI.

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Mallilankaraman, K., Doonan, P., Cárdenas, C., Chandramoorthy, H., Müller, M., Miller, R., Hoffman, N., Gandhirajan, R., Molgó, J., Birnbaum, M., Rothberg, B., Mak, D., Foskett, J. and Madesh, M. (2012). MICU1 Is an Essential Gatekeeper for MCU-Mediated Mitochondrial Ca2+ Uptake that Regulates Cell Survival. Cell, 151(3), pp.630-644.

Abstract

Ca(2+) flux across the mitochondrial inner membrane regulates bioenergetics, cytoplasmic Ca(2+) signals and activation of cell death pathways. Mitochondrial Ca(2+) uptake occurs at regions of close apposition with intracellular Ca(2+) release sites, driven by the inner membrane voltage generated by oxidative phosphorylation and mediated by a Ca(2+) selective ion channel (MiCa; ref. ) called the uniporter whose complete molecular identity remains unknown. Mitochondrial calcium uniporter (MCU) was recently identified as the likely ion-conducting pore. In addition, MICU1 was identified as a mitochondrial regulator of uniporter-mediated Ca(2+) uptake in HeLa cells. Here we identified CCDC90A, hereafter referred to as MCUR1 (mitochondrial calcium uniporter regulator 1), an integral membrane protein required for MCU-dependent mitochondrial Ca(2+) uptake. MCUR1 binds to MCU and regulates ruthenium-red-sensitive MCU-dependent Ca(2+) uptake. MCUR1 knockdown does not alter MCU localization, but abrogates Ca(2+) uptake by energized mitochondria in intact and permeabilized cells. Ablation of MCUR1 disrupts oxidative phosphorylation, lowers cellular ATP and activates AMP kinase-dependent pro-survival autophagy. Thus, MCUR1 is a critical component of a mitochondrial uniporter channel complex required for mitochondrial Ca(2+) uptake and maintenance of normal cellular bioenergetics.

Mallilankaraman, K., Doonan, P., Cárdenas, C., Chandramoorthy, H., Müller, M., Miller, R., Hoffman, N., Gandhirajan, R., Molgó, J., Birnbaum, M., Rothberg, B., Mak, D., Foskett, J. and Madesh, M. (2012). MICU1 Is an Essential Gatekeeper for MCU-Mediated Mitochondrial Ca2+ Uptake that Regulates Cell Survival. Cell, 151(3), pp.630-644.

Abstract

Ca(2+) flux across the mitochondrial inner membrane regulates bioenergetics, cytoplasmic Ca(2+) signals and activation of cell death pathways. Mitochondrial Ca(2+) uptake occurs at regions of close apposition with intracellular Ca(2+) release sites, driven by the inner membrane voltage generated by oxidative phosphorylation and mediated by a Ca(2+) selective ion channel (MiCa; ref. ) called the uniporter whose complete molecular identity remains unknown. Mitochondrial calcium uniporter (MCU) was recently identified as the likely ion-conducting pore. In addition, MICU1 was identified as a mitochondrial regulator of uniporter-mediated Ca(2+) uptake in HeLa cells. Here we identified CCDC90A, hereafter referred to as MCUR1 (mitochondrial calcium uniporter regulator 1), an integral membrane protein required for MCU-dependent mitochondrial Ca(2+) uptake. MCUR1 binds to MCU and regulates ruthenium-red-sensitive MCU-dependent Ca(2+) uptake. MCUR1 knockdown does not alter MCU localization, but abrogates Ca(2+) uptake by energized mitochondria in intact and permeabilized cells. Ablation of MCUR1 disrupts oxidative phosphorylation, lowers cellular ATP and activates AMP kinase-dependent pro-survival autophagy. Thus, MCUR1 is a critical component of a mitochondrial uniporter channel complex required for mitochondrial Ca(2+) uptake and maintenance of normal cellular bioenergetics.

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Deliu, E., Brailoiu, G., Mallilankaraman, K., Wang, H., Madesh, M., Undieh, A., Koch, W. and Brailoiu, E. (2012). Intracellular Endothelin Type B Receptor-driven Ca2+Signal Elicits Nitric Oxide Production in Endothelial Cells. Journal of Biological Chemistry, 287(49), pp.41023-41031.

Abstract

Endothelin-1 exerts its actions via activation of ET(A) and ET(B) G(q/11) protein-coupled receptors, located in the plasmalemma, cytoplasm, and nucleus. Although the autocrine/paracrine nature of endothelin-1 signaling has been extensively studied, its intracrine role has been largely attributed to interaction with receptors located on nuclear membranes and the nucleoplasm. Because ET(B) receptors have been shown to be targeted to endolysosomes, we used intracellular microinjection and concurrent imaging methods to test their involvement in Ca(2+) signaling and subsequential NO production. We provide evidence that microinjected endothelin-1 produces a dose-dependent elevation in cytosolic calcium concentration in ET(B)-transfected cells and endothelial cells; this response is sensitive to ET(B) but not ET(A) receptor blockade. In endothelial cells, the endothelin-1-induced Ca(2+) response is abolished upon endolysosomal but not Golgi disruption. Moreover, the effect is prevented by inhibition of microautophagy and is sensitive to inhibitors of the phospholipase C and inositol 1,4,5-trisphosphate receptor. Furthermore, intracellular endothelin-1 increases nitric oxide via an ET(B)-dependent mechanism. Our results indicate for the first time that intracellular endothelin-1 activates endolysosomal ET(B) receptors and increase cytosolic Ca(2+) and nitric oxide production. Endothelin-1 acts in an intracrine fashion on endolysosomal ET(B) to induce nitric oxide formation, thus modulating endothelial function.

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Mancarella, S., Wang, Y., Deng, X., Landesberg, G., Scalia, R., Panettieri, R., Mallilankaraman, K., Tang, X., Madesh, M. and Gill, D. (2011). Hypoxia-induced Acidosis Uncouples the STIM-Orai Calcium Signaling Complex. Journal of Biological Chemistry, 286(52), pp.44788-44798.

Abstract

The endoplasmic reticulum Ca(2+)-sensing STIM proteins mediate Ca(2+) entry signals by coupling to activate plasma membrane Orai channels. We reveal that STIM-Orai coupling is rapidly blocked by hypoxia and the ensuing decrease in cytosolic pH. In smooth muscle cells or HEK293 cells coexpressing STIM1 and Orai1, acute hypoxic conditions rapidly blocked store-operated Ca(2+) entry and the Orai1-mediated Ca(2+) release-activated Ca(2+) current (I(CRAC)). Hypoxia-induced blockade of Ca(2+) entry and I(CRAC) was reversed by NH(4)(+)-induced cytosolic alkalinization. Hypoxia and acidification both blocked I(CRAC) induced by the short STIM1 Orai-activating region. Although hypoxia induced STIM1 translocation into junctions, it did not dissociate the STIM1-Orai1 complex. However, both hypoxia and cytosolic acidosis rapidly decreased Förster resonance energy transfer (FRET) between STIM1-YFP and Orai1-CFP. Thus, although hypoxia promotes STIM1 junctional accumulation, the ensuing acidification functionally uncouples the STIM1-Orai1 complex providing an important mechanism protecting cells from Ca(2+) overload under hypoxic stress conditions.

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Irrinki, K., Mallilankaraman, K., Thapa, R., Chandramoorthy, H., Smith, F., Jog, N., Gandhirajan, R., Kelsen, S., Houser, S., May, M., Balachandran, S. and Madesh, M. (2011). Requirement of FADD, NEMO, and BAX/BAK for Aberrant Mitochondrial Function in Tumor Necrosis Factor Alpha-Induced Necrosis. Molecular and Cellular Biology, 31(18), pp.3745-3758.

Abstract

Necroptosis represents a form of alternative programmed cell death that is dependent on the kinase RIP1. RIP1-dependent necroptotic death manifests as increased reactive oxygen species (ROS) production in mitochondria and is accompanied by loss of ATP biogenesis and eventual dissipation of mitochondrial membrane potential. Here, we show that tumor necrosis factor alpha (TNF-α)-induced necroptosis requires the adaptor proteins FADD and NEMO. FADD was found to mediate formation of the TNF-α-induced pronecrotic RIP1-RIP3 kinase complex, whereas the IκB Kinase (IKK) subunit NEMO appears to function downstream of RIP1-RIP3. Interestingly, loss of RelA potentiated TNF-α-dependent necroptosis, indicating that NEMO regulates necroptosis independently of NF-κB. Using both pharmacologic and genetic approaches, we demonstrate that the overexpression of antioxidants alleviates ROS elevation and necroptosis. Finally, elimination of BAX and BAK or overexpression of Bcl-x(L) protects cells from necroptosis at a later step. These findings provide evidence that mitochondria play an amplifying role in inflammation-induced necroptosis.

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Cheng, Z., Jiang, X., Kruger, W., Pratico, D., Gupta, S., Mallilankaraman, K., Madesh, M., Schafer, A., Durante, W., Yang, X. and Wang, H. (2011). Hyperhomocysteinemia impairs endothelium-derived hyperpolarizing factor-mediated vasorelaxation in transgenic cystathionine beta synthase-deficient mice. Blood, 118(7), pp.1998-2006.

Abstract

Hyperhomocysteinemia (HHcy) is associated with endothelial dysfunction (ED), but the mechanism is largely unknown. In this study, we investigated the role and mechanism of HHcy-induced ED in microvasculature in our newly established mouse model of severe HHcy (plasma total homocysteine, 169.5 μM). We found that severe HHcy impaired nitric oxide (NO)- and endothelium-derived hyperpolarizing factor (EDHF)-mediated, endothelium-dependent relaxations of small mesenteric arteries (SMAs). Endothelium-independent and prostacyclin-mediated endothelium-dependent relaxations were not changed. A nonselective Ca(2+)-activated potassium channel (K(Ca)) inhibitor completely blocked EDHF-mediated relaxation. Selective blockers for small-conductance K(Ca) (SK) or intermediate-conductance K(Ca) (IK) failed to inhibit EDHF-mediated relaxation in HHcy mice. HHcy increased the levels of SK3 and IK1 protein, superoxide (O(2)(-)), and 3-nitrotyrosine in the endothelium of SMAs. Preincubation with antioxidants and peroxynitrite (ONOO(-)) inhibitors improved endothelium-dependent and EDHF-mediated relaxations and decreased O(2)(-) production in SMAs from HHcy mice. Further, EDHF-mediated relaxation was inhibited by ONOO(-) and prevented by catalase in the control mice. Finally, L-homocysteine stimulated O(2)(-) production, which was reversed by antioxidants, and increased SK/IK protein levels and tyrosine nitration in cultured human cardiac microvascular endothelial cells. Our results suggest that HHcy impairs EDHF relaxation in SMAs by inhibiting SK/IK activities via oxidation- and tyrosine nitration-related mechanisms.

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Thapa, R., Basagoudanavar, S., Nogusa, S., Irrinki, K., Mallilankaraman, K., Slifker, M., Beg, A., Madesh, M. and Balachandran, S. (2011). NF- B Protects Cells from Gamma Interferon-Induced RIP1-Dependent Necroptosis. Molecular and Cellular Biology, 31(14), pp.2934-2946.

Abstract

Interferons (IFNs) are cytokines with well-described immunomodulatory and antiviral properties, but less is known about the mechanisms by which they promote cell survival or cell death. Here, we show that IFN-γ induces RIP1 kinase-dependent necroptosis in mammalian cells deficient in NF-κB signaling. Induction of necroptosis by IFN-γ was found to depend on Jak1 and partially on STAT1. We also demonstrate that IFN-γ activates IκB kinase β (IKKβ)-dependent NF-κB to regulate a transcriptional program that protects cells from necroptosis. IFN-γ induced progressive accumulation of reactive oxygen species (ROS) and eventual loss of mitochondrial membrane potential in cells lacking the NF-κB subunit RelA. Whole-genome microarray analyses identified sod2, encoding the antioxidant enzyme manganese superoxide dismutase (MnSOD), as a RelA target and potential antinecroptotic gene. Overexpression of MnSOD inhibited IFN-γ-mediated ROS accumulation and partially rescued RelA-deficient cells from necroptosis, while RNA interference (RNAi)-mediated silencing of sod2 expression increased susceptibility to IFN-γ-induced cell death. Together, these studies demonstrate that NF-κB protects cells from IFN-γ-mediated necroptosis by transcriptionally activating a survival response that quenches ROS to preserve mitochondrial integrity.

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Mallilankaraman, K., Shedlock, D., Bao, H., Kawalekar, O., Fagone, P., Ramanathan, A., Ferraro, B., Stabenow, J., Vijayachari, P., Sundaram, S., Muruganandam, N., Sarangan, G., Srikanth, P., Khan, A., Lewis, M., Kim, J., Sardesai, N., Muthumani, K. and Weiner, D. (2011). A DNA Vaccine against Chikungunya Virus Is Protective in Mice and Induces Neutralizing Antibodies in Mice and Nonhuman Primates. PLoS Neglected Tropical Diseases, 5(1), p.e928.

Abstract

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen. While the Aedes aegypti mosquito is its primary vector, recent evidence suggests that other carriers can transmit CHIKV thus raising concerns about its spread outside of natural endemic areas to new countries including the U.S. and Europe. Considering the potential for pandemic spread, understanding the development of immunity is paramount to the development of effective counter measures against CHIKV. In this study, we isolated a new CHIKV virus from an acutely infected human patient and developed a defined viral challenge stock in mice that allowed us to study viral pathogenesis and develop a viral neutralization assay. We then constructed a synthetic DNA vaccine delivered by in vivo electroporation (EP) that expresses a component of the CHIKV envelope glycoprotein and used this model to evaluate its efficacy. Vaccination induced robust antigen-specific cellular and humoral immune responses, which individually were capable of providing protection against CHIKV challenge in mice. Furthermore, vaccine studies in rhesus macaques demonstrated induction of nAb responses, which mimicked those induced in convalescent human patient sera. These data suggest a protective role for nAb against CHIKV disease and support further study of envelope-based CHIKV DNA vaccines.

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Shedlock, D., Talbott, K., Cress, C., Ferraro, B., Tuyishme, S., Mallilankaraman, K., Cisper, N., Morrow, M., Wu, S., Kawalekar, O., Khan, A., Sardesai, N., Muthumani, K., Shen, H. and Weiner, D. (2011). A highly optimized DNA vaccine confers complete protective immunity against high-dose lethal lymphocytic choriomeningitis virus challenge. Vaccine, 29(39), pp.6755-6762.

Abstract

Protection against infection is the hallmark of immunity and the basis of effective vaccination. For a variety of reasons there is a great demand to develop new, safer and more effective vaccine platforms. In this regard, while 'first-generation' DNA vaccines were poorly immunogenic, new genetic 'optimization' strategies and the application of in vivo electroporation (EP) have dramatically boosted their potency. We developed a highly optimized plasmid DNA vaccine that expresses the lymphocytic choriomeningitis virus (LCMV) nucleocapsid protein (NP) and evaluated it using the LCMV challenge model, a gold standard for studying infection and immunity. When administered intramuscularly with EP, robust NP-specific cellular and humoral immune responses were elicited, the magnitudes of which approached those following acute LCMV infection. Furthermore, these responses were capable of providing 100% protection against a high-dose, normally lethal virus challenge. This is the first non-infectious vaccine conferring complete protective immunity up to 8 weeks after vaccination and demonstrates the potential of 'next-generation' DNA vaccines.

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Srikanth, P., Sarangan, G., Mallilankaraman, K., Nayar, S., Barani, R., Mattew, T., Selvaraj, G., Sheriff, K., Palani, G. and Muthumani, K. (2010). Molecular characterization of Chikungunya virus during an outbreak in South India. Indian Journal of Medical Microbiology, 28(4), p.299.

Abstract

INTRODUCTION: Re-emergence of Chikungunya is a major public health problem in the southern states of India.

OBJECTIVES: This study was undertaken to investigate an outbreak of Chikungunya, in June-August 2008 using PCR and determine the prevalent genotypes of Chikungunya virus (CHIKV) associated with the outbreak.

MATERIALS AND METHODS: Samples of blood were collected (in heparinized vacutainer tubes) from suspected patients of CHIKV infection from both Government Taluk Hospital in Kerala and a tertiary care hospital in Chennai, Tamil Nadu. A one-step RT-PCR was carried out on a block thermo-cycler targeting the E2 gene that codes for the viral envelope protein. The amplicons were verified for 305 bp size by standard agarose gel electrophoresis. The PCR products were purified, sequenced, and compared with other CHIKV strains reported from different geographical regions. A phylogenetic tree was constructed using MEGA 4.

RESULTS: Altogether 118 samples were collected from patients who presented with sudden onset of fever and/or joint pain, myalgia, and headache. CHIKV infection was confirmed by RT-PCR in 14 patients and all these cases were from Kerala. The positivity correlated with the early stage of the disease as all these patients had fever of less than seven days duration. The study isolates have been allotted the GenBank accession nos. GQ272368-GQ272381. Phylogenetic analysis of recent CHIKV isolates by partial sequencing of E2 region shows that isolates are closely related to strains from neighboring states and the African type.

CONCLUSION: RT-PCR is a useful technique for the early detection of CHIKV infection during outbreaks. Molecular characterization of the strains indicates that majority of the strains have originated from the Central/East African strains of CHIKV.

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Hawkins, B., Irrinki, K., Mallilankaraman, K., Lien, Y., Wang, Y., Bhanumathy, C., Subbiah, R., Ritchie, M., Soboloff, J., Baba, Y., Kurosaki, T., Joseph, S., Gill, D. and Madesh, M. (2010). S-glutathionylation activates STIM1 and alters mitochondrial homeostasis. The Journal of Cell Biology, 190(3), pp.391-405.

Abstract

Oxidant stress influences many cellular processes, including cell growth, differentiation, and cell death. A well-recognized link between these processes and oxidant stress is via alterations in Ca(2+) signaling. However, precisely how oxidants influence Ca(2+) signaling remains unclear. Oxidant stress led to a phenotypic shift in Ca(2+) mobilization from an oscillatory to a sustained elevated pattern via calcium release-activated calcium (CRAC)-mediated capacitive Ca(2+) entry, and stromal interaction molecule 1 (STIM1)- and Orai1-deficient cells are resistant to oxidant stress. Functionally, oxidant-induced Ca(2+) entry alters mitochondrial Ca(2+) handling and bioenergetics and triggers cell death. STIM1 is S-glutathionylated at cysteine 56 in response to oxidant stress and evokes constitutive Ca(2+) entry independent of intracellular Ca(2+) stores. These experiments reveal that cysteine 56 is a sensor for oxidant-dependent activation of STIM1 and demonstrate a molecular link between oxidant stress and Ca(2+) signaling via the CRAC channel.

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Thieu, K., Morrow, M., Shedlock, D., Schoenly, K., Mallilankaraman, K., Choo, A., Fagone, P., Weiner, D. and Muthumani, K. (2009). HIV-1 Vpr: Regulator of Viral Survival. Current HIV Research, 7(2), pp.153-162.

Abstract

The HIV-1 Vpr protein is a viral accessory protein that plays a number of important roles during HIV infection. The activities of Vpr are numerous and include the induction of apoptosis, the modulation of cell cycle arrest, as well as control of viral transcription. Study of HIV clones lacking Vpr in vitro and analysis of HIV variants isolated from long-term nonprogressors in vivo highlight the importance of Vpr for viral replication as well as immune suppression and cell death. Vpr may therefore be considered among the most important accessory proteins encoded by HIV.

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