Publications by authors named "Salim Merali"

65 Publications

A Novel Benzopyrane Derivative Targeting Cancer Cell Metabolic and Survival Pathways.

Cancers (Basel) 2021 Jun 7;13(11). Epub 2021 Jun 7.

Sharjah Institute for Medical Researches, University of Sharjah, Sharjah 27272, United Arab Emirates.

(1) Background: Today, the discovery of novel anticancer agents with multitarget effects and high safety margins represents a high challenge. Drug discovery efforts indicated that benzopyrane scaffolds possess a wide range of pharmacological activities. This spurs on building a skeletally diverse library of benzopyranes to identify an anticancer lead drug candidate. Here, we aim to characterize the anticancer effect of a novel benzopyrane derivative, aiming to develop a promising clinical anticancer candidate. (2) Methods: The anticancer effect of SIMR1281 against a panel of cancer cell lines was tested. In vitro assays were performed to determine the effect of SIMR1281 on GSHR, TrxR, mitochondrial metabolism, DNA damage, cell cycle progression, and the induction of apoptosis. Additionally, SIMR1281 was evaluated in vivo for its safety and in a xenograft mice model. (3) Results: SIMR1281 strongly inhibits GSHR while it moderately inhibits TrxR and modulates the mitochondrial metabolism. SIMR1281 inhibits the cell proliferation of various cancers. The antiproliferative activity of SIMR1281 was mediated through the induction of DNA damage, perturbations in the cell cycle, and the inactivation of Ras/ERK and PI3K/Akt pathways. Furthermore, SIMR1281 induced apoptosis and attenuated cell survival machinery. In addition, SIMR1281 reduced the tumor volume in a xenograft model while maintaining a high in vivo safety profile at a high dose. (4) Conclusions: Our findings demonstrate the anticancer multitarget effect of SIMR1281, including the dual inhibition of glutathione and thioredoxin reductases. These findings support the development of SIMR1281 in preclinical and clinical settings, as it represents a potential lead compound for the treatment of cancer.
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http://dx.doi.org/10.3390/cancers13112840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201054PMC
June 2021

Short chain fatty acids delay the development of hepatocellular carcinoma in HBx transgenic mice.

Neoplasia 2021 05 1;23(5):529-538. Epub 2021 May 1.

Department of Biology, College of Science and Technology, Philadelphia, PA, USA. Electronic address:

Chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The HBV encoded oncoprotein, HBx, alters the expression of host genes and the activity of multiple signal transduction pathways that contribute to the pathogenesis of HCC by multiple mechanisms independent of HBV replication. However, it is not clear which pathways are the most relevant therapeutic targets in hepatocarcinogenesis. Short chain fatty acids (SCFAs) have strong anti-inflammatory and anti-neoplastic properties, suggesting that they may block the progression of chronic liver disease (CLD) to HCC, thereby identifying the mechanisms relevant to HCC development. This hypothesis was tested in HBx transgenic (HBxTg) mice fed SCFAs. Groups of HBxTg mice were fed with SCFAs or vehicle from 6 to 9 months of age and then assessed for dysplasia, and from 9 to 12 months of age and then assessed for HCC. Livers from 12 month old mice were then analyzed for changes in gene expression by mass spectrometry-based proteomics. SCFA-fed mice had significantly fewer dysplastic and HCC nodules compared to controls at 9 and 12 months, respectively. Pathway analysis of SCFA-fed mice showed down-regulation of signaling pathways altered by HBx in human CLD and HCC, including those involved in inflammation, phosphatidylinositol 3-kinase, epidermal growth factor, and Ras. SCFA treatment promoted increased expression of the tumor suppressor, disabled homolog 2 (DAB2). DAB2 depresses Ras pathway activity, which is constitutively activated by HBx. SCFAs also reduced cell viability in HBx-transfected cell lines in a dose-dependent manner while the viability of primary human hepatocytes was unaffected. These unique findings demonstrate that SCFAs delay the pathogenesis of CLD and development of HCC, and provide insight into some of the underlying mechanisms that are relevant to pathogenesis in that they are responsive to treatment.
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http://dx.doi.org/10.1016/j.neo.2021.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8111251PMC
May 2021

A novel platform for peptide-mediated affinity capture and LC-MS/MS identification of host receptors involved in Plasmodium invasion.

J Proteomics 2021 01 9;231:104002. Epub 2020 Oct 9.

Molecular Biology and Immunology Department, Fundación Instituto de Inmunología de Colombia (FIDIC), Bogotá, Colombia; Basic Sciences Department, School of Medicine and Health Sciences, Universidad del Rosario, Bogotá, Colombia. Electronic address:

Successful Plasmodium falciparum invasion of red blood cells includes the orderly execution of highly specific receptor-ligand molecular interactions between the parasite's proteins and the red blood cell membrane proteins. There is a growing need for elucidating receptor-ligand pairings, which will help in understanding the parasite's biology and provide the fundamental basis for developing prophylactic or therapeutic alternatives leading to mitigating or eliminating this type of malaria. We have thus used Plasmodium falciparum RH5 - derived peptides and ghost red blood cell proteins in synthetic peptide affinity capture assays to identify important host receptors used by Plasmodium spp. in the invasion of red blood cells. LC-MS/MS analysis confirmed the extensively described interaction between PfRH5 and the basigin receptor on the red blood cell membrane. As shown here, tagged synthetic peptides displaying high binding ability to erythrocytes can be used to identify receptors present in protein extracts from ghost red blood cells via affinity capture and LC-MS/MS. SIGNIFICANCE: The article describes a novel approach for identifying red blood cell receptors based on the ability of synthetic peptides having high red blood cell binding capacity to capture Plasmodium spp. receptors on proteins extracted from ghost red blood cells. Specifically, novel methods to identify Plasmodium falciparum reticulocyte binding protein homolog 5 PfRH5 and basigin interaction using a combination of affinity capture and LC-MS/MS assays is described. Identification of these host RBC receptors interacting with malarial parasite proteins is of utmost importance in studying the disease's pathogenesis and will provide crucial information in understanding the parasite's biology. In addition, data from these studies can be used to identify potential therapeutic target(s) to mitigate or eliminate this debilitating disease.
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http://dx.doi.org/10.1016/j.jprot.2020.104002DOI Listing
January 2021

Transient receptor potential ion channel TRPM2 promotes AML proliferation and survival through modulation of mitochondrial function, ROS, and autophagy.

Cell Death Dis 2020 04 20;11(4):247. Epub 2020 Apr 20.

Department of Pediatrics, The Pennsylvania State University College of Medicine, P.O. Box 850, Hershey, PA, 17033, USA.

Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in maintaining cell survival following oxidant injury. Here, we show that TRPM2 is highly expressed in acute myeloid leukemia (AML). The role of TRPM2 in AML was studied following depletion with CRISPR/Cas9 technology in U937 cells. In in vitro experiments and in xenografts, depletion of TRPM2 in AML inhibited leukemia proliferation, and doxorubicin sensitivity was increased. Mitochondrial function including oxygen consumption rate and ATP production was reduced, impairing cellular bioenergetics. Mitochondrial membrane potential and mitochondrial calcium uptake were significantly decreased in depleted cells. Mitochondrial reactive oxygen species (ROS) were significantly increased, and Nrf2 was decreased, reducing the antioxidant response. In TRPM2-depleted cells, ULK1, Atg7, and Atg5 protein levels were decreased, leading to autophagy inhibition. Consistently, ATF4 and CREB, two master transcription factors for autophagosome biogenesis, were reduced in TRPM2-depleted cells. In addition, Atg13 and FIP200, which are known to stabilize ULK1 protein, were decreased. Reconstitution with TRPM2 fully restored proliferation, viability, and autophagy; ATF4 and CREB fully restored proliferation and viability but only partially restored autophagy. TRPM2 expression reduced the elevated ROS found in depleted cells. These data show that TRPM2 has an important role in AML proliferation and survival through regulation of key transcription factors and target genes involved in mitochondrial function, bioenergetics, the antioxidant response, and autophagy. Targeting TRPM2 may represent a novel therapeutic approach to inhibit myeloid leukemia growth and enhance susceptibility to chemotherapeutic agents through multiple pathways.
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http://dx.doi.org/10.1038/s41419-020-2454-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170900PMC
April 2020

The central role of protein kinase C epsilon in cyanide cardiotoxicity and its treatment.

Toxicol Sci 2019 Jun 7. Epub 2019 Jun 7.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Pennsylvania State University College of Medicine, Hershey, PA.

In adult mouse myocytes, brief exposure to sodium cyanide (CN) in the presence of glucose does not decrease ATP levels, yet produces profound reduction in contractility, intracellular Ca2+ concentration ([Ca2+]i) transient and L-type Ca2+ current (ICa) amplitudes. We analyzed proteomes from myocytes exposed to CN, focusing on ionic currents associated with excitation-contraction coupling. CN induced phosphorylation of α1c subunit of L-type Ca2+ channel and α2 subunit of Na+-K+-ATPase. Methylene blue (MB), a CN antidote that we previously reported to ameliorate CN-induced reduction in contraction, [Ca2+]i transient and ICa amplitudes, was able to reverse this phosphorylation. CN decreased Na+-K+-ATPase current contributed by α2 but not α1 subunit, an effect that was also counteracted by MB. Peptide consensus sequences suggested CN-induced phosphorylation was mediated by protein kinase C epsilon (PKCε). Indeed, CN stimulated PKC kinase activity and induced PKCε membrane translocation, effects that were prevented by MB. Pre-treatment with myristoylated PKCε translocation activator or inhibitor peptides mimicked and inhibited the effects of CN on ICa and myocyte contraction, respectively. We conclude that CN activates PKCε, which phosphorylates L-type Ca2+ channel and Na+-K+-ATPase, resulting in depressed cardiac contractility. We hypothesize that this inhibition of ion fluxes represents a novel mechanism by which the cardiomyocyte reduces its ATP demand (decreased ion fluxes and contractility), diminishes ATP turnover and preserves cell viability. However, this cellular protective effect translates into life-threatening cardiogenic shock in vivo, thereby creating a profound disconnect between survival mechanisms at the cardiomyocyte level from those at the level of the whole organism.
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http://dx.doi.org/10.1093/toxsci/kfz137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735853PMC
June 2019

Novel biomarkers to assess in utero effects of maternal opioid use: First steps toward understanding short- and long-term neurodevelopmental sequelae.

Genes Brain Behav 2019 07 11;18(6):e12583. Epub 2019 Jun 11.

Department of Psychology, Temple University, Philadelphia, Pennsylvania.

Maternal opioid use disorder is common, resulting in significant neonatal morbidity and cost. Currently, it is not possible to predict which opioid-exposed newborns will require pharmacotherapy for neonatal abstinence syndrome. Further, little is known regarding the effects of maternal opioid use disorder on the developing human brain. We hypothesized that novel methodologies utilizing fetal central nervous system-derived extracellular vesicles isolated from maternal blood can address these gaps in knowledge. Plasma from opioid users and controls between 9 and 21 weeks was precipitated and extracellular vesicles were isolated. Mu opioid and cannabinoid receptor levels were quantified. Label-free proteomics studies and unbiased small RNA next generation sequencing was performed in paired fetal brain tissue. Maternal opioid use disorder increased mu opioid receptor protein levels in extracellular vesicles independent of opioid equivalent dose. Moreover, cannabinoid receptor levels in extracellular vesicles were upregulated with opioid exposure indicating cross talk with endocannabinoids. Maternal opioid use disorder was associated with significant changes in extracellular vesicle protein cargo and fetal brain micro RNA expression, especially in male fetuses. Many of the altered cargo molecules and micro RNAs identified are associated with adverse clinical neurodevelopmental outcomes. Our data suggest that assays relying on extracellular vesicles isolated from maternal blood extracellular vesicles may provide information regarding fetal response to opioids in the setting of maternal opioid use disorder. Prospective clinical studies are needed to evaluate the association between extracellular vesicle biomarkers, risk of neonatal abstinence syndrome and neurodevelopmental outcomes.
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http://dx.doi.org/10.1111/gbb.12583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074845PMC
July 2019

ELMO1 deficiency enhances platelet function.

Blood Adv 2019 02;3(4):575-587

Department of Physiology and.

Phosphatidylinositol 3-kinase is an important signaling molecule that, once activated, leads to the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP). We performed a proteomic screen to identify PIP-interacting proteins in human platelets Among these proteins, we found engulfment and cell motility 1 (ELMO1), a scaffold protein with no catalytic activity. ELMO1 is expressed in platelets and interacts with active RhoG. However, the function of ELMO1 in platelets is not known. The focus of this study was to determine the function of ELMO1 in platelets utilizing ELMO1 mice. Platelet aggregation, granule secretion, integrin αIIbβ3 activation, and thromboxane generation were enhanced in ELMO1 platelets in response to glycoprotein VI (GPVI) agonists but unaltered when a protease-activated receptor 4 agonist was used. The kinetics of spreading on immobilized fibrinogen was enhanced in ELMO1 platelets compared with wild-type (WT) littermate controls. This suggests that ELMO1 plays a role downstream of the GPVI and integrin αIIbβ3 pathway. Furthermore, whole blood from ELMO1 mice perfused over collagen exhibited enhanced thrombus formation compared with WT littermate controls. ELMO1 mice showed reduced survival compared with control following pulmonary embolism. ELMO1 mice also exhibited a shorter time to occlusion using the ferric-chloride injury model and reduced bleeding times compared with WT littermate controls. These results indicate that ELMO1 plays an important role in hemostasis and thrombosis in vivo. RhoG activity was enhanced in ELMO1 murine platelets compared with WT littermate controls in response to GPVI agonist. Together, these data suggest that ELMO1 negatively regulates GPVI-mediated thrombus formation via RhoG.
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http://dx.doi.org/10.1182/bloodadvances.2018016444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391667PMC
February 2019

Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cell 2018 11 25;175(5):1244-1258.e26. Epub 2018 Oct 25.

The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
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http://dx.doi.org/10.1016/j.cell.2018.09.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247954PMC
November 2018

FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation.

Nat Commun 2018 08 29;9(1):3449. Epub 2018 Aug 29.

Department of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, 19140, USA.

Although many factors contribute to cellular differentiation, the role of mitochondria Ca dynamics during development remains unexplored. Because mammalian embryonic epiblasts reside in a hypoxic environment, we intended to understand whether Ca and its transport machineries are regulated during hypoxia. Tissues from multiple organs of developing mouse embryo evidenced a suppression of MICU1 expression with nominal changes on other MCU complex components. As surrogate models, we here utilized human embryonic stem cells (hESCs)/induced pluripotent stem cells (hiPSCs) and primary neonatal myocytes to delineate the mechanisms that control Ca and bioenergetics during development. Analysis of MICU1 expression in hESCs/hiPSCs showed low abundance of MICU1 due to its direct repression by Foxd1. Experimentally, restoration of MICU1 established the periodic Ca oscillations and promoted cellular differentiation and maturation. These findings establish a role of Ca dynamics in regulation of cellular differentiation and reveal a molecular mechanism underlying this contribution through differential regulation of MICU1.
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http://dx.doi.org/10.1038/s41467-018-05856-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115453PMC
August 2018

Elevated levels of brain homocysteine directly modulate the pathological phenotype of a mouse model of tauopathy.

Mol Psychiatry 2019 11 4;24(11):1696-1706. Epub 2018 May 4.

Alzheimer's Center at Temple, Lewis Katz School of Medicine, Temple University, Philadelphia, PA, 19140, USA.

A high circulating level of homocysteine (Hcy), also known as hyperhomocysteinemia, is a risk factor for Alzheimer's disease (AD). Previous studies show that elevated Hcy promotes brain amyloidosis and behavioral deficits in mouse models of AD. However, whether it directly modulates the development of tau neuropathology independently of amyloid beta in vivo is unknown. Herein, we investigate the effect of diet-induced elevated levels of brain Hcy on the phenotype of a relevant mouse model of human tauopathy. Compared with controls, tau mice fed with low folate and B vitamins diet had a significant increase in brain Hcy levels and worsening of behavioral deficits. The same mice had a significant elevation of tau phosphorylation, synaptic pathology, and astrocytes activation. In vitro studies demonstrated that Hcy effect on tau phosphorylation was mediated by an upregulation of 5-lipoxygenase via cdk5 kinase pathway activation. Our findings support the novel concept that high Hcy level in the central nervous system is a metabolic risk factor for neurodegenerative diseases, specifically characterized by the progressive accumulation of tau pathology, namely tauopathies.
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http://dx.doi.org/10.1038/s41380-018-0062-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215750PMC
November 2019

Pharmacological stimulation of nuclear factor (erythroid-derived 2)-like 2 translation activates antioxidant responses.

J Biol Chem 2017 08 6;292(34):14108-14121. Epub 2017 Jul 6.

From the Department of Pharmaceutical Sciences, Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, Philadelphia, Pennsylvania 19140. Electronic address:

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the master regulator of the antioxidant response, and its function is tightly regulated at the transcriptional, translational, and post-translational levels. It is well-known that Nrf2 is regulated at the protein level by proteasomal degradation via Kelch-like ECH-associated protein 1 (Keap1), but how Nrf2 is regulated at the translational level is less clear. Here, we show that pharmacological stimulation increases Nrf2 levels by overcoming basal translational repression. We developed a novel reporter assay that enabled identification of natural compounds that induce Nrf2 translation by a mechanism independent of Keap1-mediated degradation. Apigenin, resveratrol, and piceatannol all induced Nrf2 translation. More importantly, the pharmacologically induced Nrf2 overcomes Keap1 regulation, translocates to the nucleus, and activates the antioxidant response. We conclude that translational regulation controls physiological levels of Nrf2, and this can be modulated by apigenin, resveratrol, and piceatannol. Also, targeting this mechanism with novel compounds could provide new insights into prevention and treatment of multiple diseases in which oxidative stress plays a significant role.
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http://dx.doi.org/10.1074/jbc.M116.770925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572918PMC
August 2017

Secretion of the endoplasmic reticulum stress protein, GRP78, into the BALF is increased in cigarette smokers.

Respir Res 2017 05 2;18(1):78. Epub 2017 May 2.

Department of Thoracic Medicine and Surgery, Temple University School of Medicine, Philadelphia, PA, 19140, USA.

Background: Identification of biomarkers of cigarette smoke -induced lung damage and early COPD is an area of intense interest. Glucose regulated protein of 78 kD (i.e., GRP78), a multi-functional protein which mediates cell responses to oxidant stress, is increased in the lungs of cigarette smokers and in the serum of subjects with COPD. We have suggested that secretion of GRP78 by lung cells may explain the increase in serum GRP78 in COPD. To assess GRP78 secretion by the lung, we assayed GRP78 in bronchoalveolar lavage fluid (BALF) in chronic smokers and non-smokers. We also directly assessed the acute effect of cigarette smoke material on GRP78 secretion in isolated human airway epithelial cells (HAEC).

Methods: GRP78 was measured in BALF of smokers (S; n = 13) and non-smokers (NS; n = 11) by Western blotting. GRP78 secretion by HAEC was assessed by comparing its concentration in cell culture medium and cell lysates. Cells were treated for 24 h with either the volatile phase of cigarette smoke (cigarette smoke extract (CSE) or the particulate phase (cigarette smoke condensate (CSC)).

Results: GRP78 was present in the BALF of both NS and S but levels were significantly greater in S (p = 0.04). GRP78 was secreted constitutively in HAEC. CSE 15% X 24 h increased GRP78 in cell-conditioned medium without affecting its intracellular concentration. In contrast, CSC X 24 h increased intracellular GRP78 expression but did not affect GRP78 secretion. Brefeldin A, an inhibitor of classical Golgi secretion pathways, did not inhibit GRP78 secretion indicating that non-classical pathways were involved.

Conclusion: The present study indicates that GRP78 is increased in BALF in cigarette smokers; that HAEC secrete GRP78; and that GRP78 secretion by HAEC is augmented by cigarette smoke particulates. Enhanced secretion of GRP78 by lung cells makes it a potential biomarker of cigarette smoke-induced lung injury.
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http://dx.doi.org/10.1186/s12931-017-0561-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414124PMC
May 2017

Five lipoxygenase hypomethylation mediates the homocysteine effect on Alzheimer's phenotype.

Sci Rep 2017 04 6;7:46002. Epub 2017 Apr 6.

Department of Pharmacology and Center for Translational Medicine, Lewis Katz School of Medicine, Temple University Philadelphia, PA 19140, USA.

Environmental and genetic risk factors are implicated in the pathogenesis of Alzheimer's disease (AD). However, how they interact and influence its pathogenesis remains to be investigated. High level of homocysteine (Hcy) is an AD risk factor and associates with an up-regulation of the ALOX5 gene. In the current paper we investigated whether this activation is responsible for the Hcy effect on the AD phenotype and the mechanisms involved. Triple transgenic mice were randomized to receive regular chow diet, a diet deficient in folate and B vitamins (Diet), which results in high Hcy, or the Diet plus zileuton, a specific ALOX5 inhibitor, for 7 months. Compared with controls, Diet-fed mice had a significant increase in Hcy levels, memory and learning deficits, up-regulation of the ALOX5 pathway, increased Aβ levels, tau phosphorylation, and synaptic pathology, which were absent in mice treated with zileuton. In vivo and vitro studies demonstrated that the mechanism responsible was the hypomethylation of the ALOX5 promoter. Our findings demonstrate that the up-regulation of the ALOX5 is responsible for the Hcy-dependent worsening of the AD phenotype in a relevant mouse model of the disease. The discovery of this previously unknown cross-talk between these two pathways could afford novel therapeutic opportunities for treating or halting AD.
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http://dx.doi.org/10.1038/srep46002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382538PMC
April 2017

Genetic absence of ALOX5 protects from homocysteine-induced memory impairment, tau phosphorylation and synaptic pathology.

Hum Mol Genet 2017 05;26(10):1855-1862

Department of Pharmacology and Center for Translational Medicine, Lewis Katz School of Medicine.

Elevated level of homocysteine (Hcy) is considered a risk factor for neurodegenerative diseases, but the mechanisms remain to be established. Because high Hcy is associated with an up-regulation of the ALOX5 gene product, the 5Lipoxygenase (5LO), herein we investigated whether this activation is responsible for the Hcy effect on neurodegeneration or is a secondary event. To reach this goal, wild type mice and mice genetically deficient for 5LO were assessed after being exposed to a diet known to significantly increase brain levels of Hcy. Confirming compliance with the dietary regimen, we found that by the end of the study brain levels of Hcy were significantly increase in both groups. However, diet-induced high Hcy resulted in a significant increase in Aβ, tau phosphorylation, neuroinflammation, synaptic pathology and memory impairment in control mice, but not in mice lacking ALOX5.Taken together our findings demonstrate that the up-regulation of the ALOX5 gene pathway is responsible for the development of the biochemical and behavioral sequelae of high Hcy brain levels in the context of a neurodegenerative phenotype. They provide critical support that this gene and its expressed protein are viable therapeutic targets to prevent the onset, or delay neurodegenerative events in subjects exposed to this risk factor.
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http://dx.doi.org/10.1093/hmg/ddx088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075360PMC
May 2017

Homocysteine modulates 5-lipoxygenase expression level via DNA methylation.

Aging Cell 2017 04 29;16(2):273-280. Epub 2016 Nov 29.

Department of Pharmacology and Center for Translational Medicine, Lewis Katz School of Medicine, Philadelphia, PA, 19140, USA.

Elevated levels of homocysteinemia (Hcy), a risk factor for late-onset Alzheimer's disease (AD), have been associated with changes in cell methylation. Alzheimer's disease is characterized by an upregulation of the 5-lipoxygenase (5LO), whose promoter is regulated by methylation. However, whether Hcy activates 5LO enzymatic pathway by influencing the methylation status of its promoter remains unknown. Brains from mice with high Hcy were assessed for the 5LO pathway and neuronal cells exposed to Hcy implemented to study the mechanism(s) regulating 5LO expression levels and the effect on amyloid β formation. Diet- and genetically induced high Hcy resulted in 5LO protein and mRNA upregulation, which was associated with a significant increase of the S-adenosylhomocysteine (SAH)/S-adenosylmethionine ratio, and reduced DNA methyltrasferases and hypomethylation of 5-lipoxygenase DNA. In vitro studies confirmed these results and demonstrated that the mechanism involved in the Hcy-dependent 5LO activation and amyloid β formation is DNA hypomethylation secondary to the elevated levels of SAH. Taken together these findings represent the first demonstration that Hcy directly influences 5LO expression levels and establish a previously unknown cross talk between these two pathways, which is highly relevant for AD pathogenesis. The discovery of such a novel link not only provides new mechanistic insights in the neurobiology of Hcy, but most importantly new therapeutic opportunities for the individuals bearing this risk factor for the disease.
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http://dx.doi.org/10.1111/acel.12550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5334532PMC
April 2017

Glutamate metabolism in HIV-1 infected macrophages: Role of HIV-1 Vpr.

Cell Cycle 2016 Sep 31;15(17):2288-98. Epub 2016 May 31.

a Department of Neuroscience and Comprehensive NeuroAIDS Center , Lewis Katz School of Medicine at Temple University , Philadelphia , PA , USA.

HIV-1 infected macrophages play a significant role in the neuropathogenesis of AIDS. HIV-1 viral protein R (Vpr) not only facilitates HIV-1 infection but also contribute to long-lived persistence in macrophages. Our previous studies using SILAC-based proteomic analysis showed that the expression of critical metabolic enzymes in the glycolytic pathway and tricarboxylic acid (TCA) cycle were altered in response to Vpr expression in macrophages. We hypothesized that Vpr-induced modulation of glycolysis and TCA cycle regulates glutamate metabolism and release in HIV-1 infected macrophages. We assessed the amount of specific metabolites induced by Vpr and HIV-1 in macrophages at the intracellular and extracellular level in a time-dependent manner utilizing multiple reaction monitoring (MRM) targeted metabolomics. In addition, stable isotope-labeled glucose and an MRM targeted metabolomics assay were used to evaluate the de novo synthesis and release of glutamate in Vpr overexpressing macrophages and HIV-1 infected macrophages, throughout the metabolic flux of glycolytic pathway and TCA cycle activation. The metabolic flux studies demonstrated an increase in glucose uptake, glutamate release and accumulation of α-ketoglutarate (α-KG) and glutamine in the extracellular milieu in Vpr expressing and HIV-1 infected macrophages. Interestingly, glutamate pools and other intracellular intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), citrate, malate, α-KG, and glutamine) showed a decreased trend except for fumarate, in contrast to the glutamine accumulation observed in the extracellular space in Vpr overexpressing macrophages. Our studies demonstrate that dysregulation of mitochondrial glutamate metabolism induced by Vpr in HIV-1 infected macrophages commonly seen, may contribute to neurodegeneration via excitotoxic mechanisms in the context of NeuroAIDS.
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http://dx.doi.org/10.1080/15384101.2016.1190054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004675PMC
September 2016

MCUR1 Is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics.

Cell Rep 2016 05 12;15(8):1673-85. Epub 2016 May 12.

Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, PA 19140, USA.

Mitochondrial Ca(2+) Uniporter (MCU)-dependent mitochondrial Ca(2+) uptake is the primary mechanism for increasing matrix Ca(2+) in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here, we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1 (MCUR1) have severely impaired [Ca(2+)]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation, and migration but elicited autophagy. These studies establish the existence of a MCU complex that assembles at the mitochondrial integral membrane and regulates Ca(2+)-dependent mitochondrial metabolism.
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http://dx.doi.org/10.1016/j.celrep.2016.04.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4880542PMC
May 2016

Disruption of NAD(+) binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions.

World J Biol Chem 2015 Nov;6(4):366-78

Manali Phadke, Natalia Krynetskaia, Carlos Barrero, Salim Merali, Scott A Gothe, Evgeny Krynetskiy, Temple University School of Pharmacy, Philadelphia, PA 19140, United States.

Aim: To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents.

Methods: We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD(+) cofactor binding.

Results: Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD(+) binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD(+) binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD(+) (Km = 741 ± 257 μmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD(+) binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD(+) binding center of GAPDH abrogated its intranuclear interactions.

Conclusion: Our results suggest an important functional role of phosphorylated amino acids in the NAD(+) binding center in GAPDH interactions with its intranuclear partners.
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http://dx.doi.org/10.4331/wjbc.v6.i4.366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4657119PMC
November 2015

Excessive caloric intake acutely causes oxidative stress, GLUT4 carbonylation, and insulin resistance in healthy men.

Sci Transl Med 2015 Sep;7(304):304re7

Department of Pharmaceutical Sciences and Moulder Center for Drug Discovery, Proteomics/Metabolomics Facility, Temple University School of Pharmacy, Philadelphia, PA 19140, USA.

Obesity-linked insulin resistance greatly increases the risk for type 2 diabetes, hypertension, dyslipidemia, and non-alcoholic fatty liver disease, together known as the metabolic or insulin resistance syndrome. How obesity promotes insulin resistance remains incompletely understood. Plasma concentrations of free fatty acids and proinflammatory cytokines, endoplasmic reticulum ( ER) stress, and oxidative stress are all elevated in obesity and have been shown to induce insulin resistance. However, they may be late events that only develop after chronic excessive nutrient intake. The nature of the initial event that produces insulin resistance at the beginning of excess caloric intake and weight gain remains unknown. We show that feeding healthy men with ~6000 kcal/day of the common U.S. diet [~50% carbohydrate (CHO), ~ 35% fat, and ~15% protein] for 1 week produced a rapid weight gain of 3.5 kg and the rapid onset (after 2 to 3 days) of systemic and adipose tissue insulin resistance and oxidative stress but no inflammatory or ER stress. In adipose tissue, the oxidative stress resulted in extensive oxidation and carbonylation of numerous proteins, including carbonylation of GLUT4 near the glucose transport channel, which likely resulted in loss of GLUT4 activity. These results suggest that the initial event caused by overnutrition may be oxidative stress, which produces insulin resistance, at least in part, via carbonylation and oxidation-induced inactivation of GLUT4.
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http://dx.doi.org/10.1126/scitranslmed.aac4765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600191PMC
September 2015

SILAC-Based Mass Spectrometry Analysis Reveals That Epibrassinolide Induces Apoptosis via Activating Endoplasmic Reticulum Stress in Prostate Cancer Cells.

PLoS One 2015 9;10(9):e0135788. Epub 2015 Sep 9.

Istanbul Kultur University, Department of Molecular Biology and Genetics, Atakoy Campus, Bakirkoy, Istanbul-Turkey.

Epibrassinolide (EBR) is a polyhydroxylated sterol derivative and biologically active compound of the brassinosteroids. In addition to well-described roles in plant growth, EBR induces apoptosis in the LNCaP prostate cancer cells expressing functional androgen receptor (AR). Therefore, it is suggested that EBR might have an inhibitory potential on androgen receptor signaling pathway. However, the mechanism by which EBR exerts its effects on LNCaP is poorly understood. To address this gap in knowledge, we used an unbiased global proteomics approach, i.e., stable-isotope labeling by amino acids in cell culture (SILAC). In total, 964 unique proteins were identified, 160 of which were differentially expressed after 12 h of EBR treatment. The quantification of the differentially expressed proteins revealed that the expression of the unfolded protein response (UPR) chaperone protein, calreticulin (CALR), was dramatically downregulated. The decrease in CALR expression was also validated by immunoblotting. Because our data revealed the involvement of the UPR in response to EBR exposure, we evaluated the expression of the other UPR proteins. We demonstrated that EBR treatment downregulated calnexin and upregulated BiP and IRE1α expression levels and induced CHOP translocation from the cytoplasm to nucleus. The translocation of CHOP was associated with caspase-9 and caspase-3 activation after a 12 h EBR treatment. Co-treatment of EBR with rapamycin, an upstream mTOR pathway inhibitor, prevented EBR-induced cell viability loss and PARP cleavage in LNCaP prostate cancer cells, suggesting that EBR could induce ER stress in these cells. In addition, we observed similar results in DU145 cells with nonfunctional androgen receptor. When proteasomal degradation of proteins was blocked by MG132 co-treatment, EBR treatment further induced PARP cleavage relative to drug treatment alone. EBR also induced Ca2+ sequestration, which confirmed the alteration of the ER pathway due to drug treatment. Therefore, we suggest that EBR promotes ER stress and induces apoptosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135788PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564160PMC
June 2016

Changes in protein expression in two cholangiocarcinoma cell lines undergoing formation of multicellular tumor spheroids in vitro.

PLoS One 2015 10;10(3):e0118906. Epub 2015 Mar 10.

Istituto Pasteur, Fondazione Cenci Bolognetti and Department of Biochemical Sciences "A. Rossi Fanelli", La Sapienza University of Rome and CNR, Biology and Molecular Pathology Institutes, Rome, Italy.

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and resistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutaminase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The arising protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conversion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the carbohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0118906PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4355290PMC
January 2016

A splice variant of the human ion channel TRPM2 modulates neuroblastoma tumor growth through hypoxia-inducible factor (HIF)-1/2α.

J Biol Chem 2014 Dec 12;289(52):36284-302. Epub 2014 Nov 12.

From the Departments of Pediatrics, Biochemistry and Molecular Biology,

The calcium-permeable ion channel TRPM2 is highly expressed in a number of cancers. In neuroblastoma, full-length TRPM2 (TRPM2-L) protected cells from moderate oxidative stress through increased levels of forkhead box transcription factor 3a (FOXO3a) and superoxide dismutase 2. Cells expressing the dominant negative short isoform (TRPM2-S) had reduced FOXO3a and superoxide dismutase 2 levels, reduced calcium influx in response to oxidative stress, and enhanced reactive oxygen species, leading to decreased cell viability. Here, in xenografts generated with SH-SY5Y neuroblastoma cells stably expressing TRPM2 isoforms, growth of tumors expressing TRPM2-S was significantly reduced compared with tumors expressing TRPM2-L. Expression of hypoxia-inducible factor (HIF)-1/2α was significantly reduced in TRPM2-S-expressing tumor cells as was expression of target proteins regulated by HIF-1/2α including those involved in glycolysis (lactate dehydrogenase A and enolase 2), oxidant stress (FOXO3a), angiogenesis (VEGF), mitophagy and mitochondrial function (BNIP3 and NDUFA4L2), and mitochondrial electron transport chain activity (cytochrome oxidase 4.1/4.2 in complex IV). The reduction in HIF-1/2α was mediated through both significantly reduced HIF-1/2α mRNA levels and increased levels of von Hippel-Lindau E3 ligase in TRPM2-S-expressing cells. Inhibition of TRPM2-L by pretreatment with clotrimazole or expression of TRPM2-S significantly increased sensitivity of cells to doxorubicin. Reduced survival of TRPM2-S-expressing cells after doxorubicin treatment was rescued by gain of HIF-1 or -2α function. These data suggest that TRPM2 activity is important for tumor growth and for cell viability and survival following doxorubicin treatment and that interference with TRPM2-L function may be a novel approach to reduce tumor growth through modulation of HIF-1/2α, mitochondrial function, and mitophagy.
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http://dx.doi.org/10.1074/jbc.M114.620922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4276889PMC
December 2014

Homocysteine exacerbates β-amyloid pathology, tau pathology, and cognitive deficit in a mouse model of Alzheimer disease with plaques and tangles.

Ann Neurol 2014 Jun 28;75(6):851-63. Epub 2014 May 28.

Department of Pharmacology and Center for Translational Medicine, School of Medicine, Temple University, Philadelphia, PA.

Objective: High level of homocysteine (Hcy) is a recognized risk factor for developing Alzheimer disease (AD). However, the mechanisms involved are unknown. Previously, it was shown that high Hcy increases brain β-amyloid (Aβ) levels in amyloid precursor protein transgenic mice, but no data are available on the effect that it may have on the other main pathologic features of AD such as tau.

Methods: 3xTg mice with diet-induced high Hcy were compared with mice having normal Hcy. Neuronal cells were incubated with and without Hcy.

Results: Diet-induced high Hcy resulted in an exacerbation of the entire AD-like phenotype of the 3xTg mice. In particular, we found that compared with controls, mice with high Hcy developed significant memory and learning deficits, and had elevated Aβ levels and deposition, which was mediated by an activation of the γ-secretase pathway. In addition, the same mice had a significant increase in the insoluble fraction of tau and its phosphorylation at specific epitopes, which was mediated by the cdk5 pathway. In vitro studies confirmed these observations and provided evidence that the effects of Hcy on Aβ and tau are independent from each other.

Interpretation: Taken together, our findings demonstrate that a dietary condition that leads to an elevation of Hcy levels results in an exacerbation of all 3 major pathological features of the AD phenotype: memory deficits, and Aβ and tau neuropathology. They support the concept that this dietary lifestyle can act as a risk factor and actively contribute to the development of the disease.
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http://dx.doi.org/10.1002/ana.24145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362695PMC
June 2014

TRPM2 channels protect against cardiac ischemia-reperfusion injury: role of mitochondria.

J Biol Chem 2014 Mar 3;289(11):7615-29. Epub 2014 Feb 3.

From the Center of Translational Medicine.

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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http://dx.doi.org/10.1074/jbc.M113.533851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3953274PMC
March 2014

Polyamines: Predictive Biomarker for HIV-Associated Neurocognitive Disorders.

J AIDS Clin Res 2014;5(6):1000312

Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA.

Objectives: Spermidine/spermine-N-acetytransferase (SSAT) is the key enzyme in the catabolism of polyamines that are involved in regulating NMDA functioning. Over expression of SSAT leads to abnormal metabolic cycling and may disrupt NMDA receptor signaling. In fact, the HIV protein Tat induces neurotoxicity involving polyamine/NMDA receptor interactions. Thus, we investigated abnormal polyamine cycling in HIV+ participants with varying degrees of HIV-associated neurocognitive disorders.

Methods: Acetyl-polyamine (SSAT products) levels were assessed by HPLC in CSF from 99 HIV-infected participants (no cognitive impairment (NCI, n=25), asymptomatic neurocognitive impairment (ANI, n=25), mild cognitive and motor disorders (MCMD, n=24), and HIV-associated dementia (HAD, n=25)). Polyamine levels in brain tissues from a subset of participants (uninfected (n=3), NCI (n=3), and MNCD (n=3)) were also assessed. Human primary astrocytes expressing HIV Tat were assessed for levels of the SSAT activity.

Results: Activation of the polyamine catabolic enzyme, SSAT increases polyamine flux in brain and CSF of HIV infected individuals with HIV-associated neurocognitive disorders. CSF levels of acetylated polyamine increase with the degree of HAND severity as indicated by significantly increased acetylpolyamine levels in HAD participants compared to NCI and ANI (p<0.0001) and between MCMD and NCI and ANI (p<0.0001). studies suggest that the HIV protein Tat may be responsible in part for astrocyte-derived acetyl polyamine release.

Interpretation: Our data suggest that polyamine metabolism may play a pivotal role in the neurodegeneration process among HAND patients. Changes in polyamine flux may serve as a potential predictive diagnostic biomarker for different severities of HAND.
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http://dx.doi.org/10.4172/2155-6113.1000312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4397651PMC
January 2014

Insulin regulates the unfolded protein response in human adipose tissue.

Diabetes 2014 Mar 15;63(3):912-22. Epub 2013 Oct 15.

Division of Endocrinology/Diabetes/Metabolism, Temple University School of Medicine, Philadelphia, PA.

Endoplasmic reticulum (ER) stress is increased in obesity and is postulated to be a major contributor to many obesity-related pathologies. Little is known about what causes ER stress in obese people. Here, we show that insulin upregulated the unfolded protein response (UPR), an adaptive reaction to ER stress, in vitro in 3T3-L1 adipocytes and in vivo, in subcutaneous (sc) adipose tissue of nondiabetic subjects, where it increased the UPR dose dependently over the entire physiologic insulin range (from ∼ 35 to ∼ 1,450 pmol/L). The insulin-induced UPR was not due to increased glucose uptake/metabolism and oxidative stress. It was associated, however, with increased protein synthesis, with accumulation of ubiquitination associated proteins, and with multiple posttranslational protein modifications (acetylations, methylations, nitrosylations, succinylation, and ubiquitinations), some of which are potential causes for ER stress. These results reveal a new physiologic role of insulin and provide a putative mechanism for the development of ER stress in obesity. They may also have clinical and therapeutic implications, e.g., in diabetic patients treated with high doses of insulin.
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http://dx.doi.org/10.2337/db13-0906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931405PMC
March 2014

Analysis of the plasma proteome in COPD: Novel low abundance proteins reflect the severity of lung remodeling.

COPD 2014 Apr 10;11(2):177-89. Epub 2013 Oct 10.

1Department of Biochemistry, Temple University School of Medicine , Philadelphia, PA , USA.

The search for COPD biomarkers has largely employed a targeted approach that focuses on plasma proteins involved in the systemic inflammatory response and in lung injury and repair. This proof of concept study was designed to test the idea that an open, unbiased, in-depth proteomics approach could identify novel, low abundance plasma proteins i.e., ng/mL concentration, which could serve as potential biomarkers. Differentially expressed proteins were identified in a discovery group with severe COPD (FEV1 <45% predicted; n = 10). Subjects with normal lung function matched for age, sex, ethnicity and smoking history served as controls (n = 10). Pooled plasma from each group was exhaustively immunodepleted of abundant proteins, d separated by 1-D gel electrophoresis and extensively fractionated prior to LC-tandem mass spectroscopy (GeLC-MS). Thirty one differentially expressed proteins were identified in the discovery group including markers of lung defense against oxidant stress, alveolar macrophage activation, and lung tissue injury and repair. Four of the 31 proteins (i.e., GRP78, soluble CD163, IL1AP and MSPT9) were measured in a separate verification group of 80 subjects with varying COPD severity by immunoassay. All 4 were significantly altered in COPD and 2 (GRP78 and soluble CD163) correlated with both FEV1 and the extent of emphysema. In-depth, plasma proteomic analysis identified a group of novel, differentially expressed, low abundance proteins that reflect known pathogenic mechanisms and the severity of lung remodeling in COPD. These proteins may also prove useful as COPD biomarkers.
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http://dx.doi.org/10.3109/15412555.2013.831063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497366PMC
April 2014

Cryptosporidium parvum induces an endoplasmic stress response in the intestinal adenocarcinoma HCT-8 cell line.

J Biol Chem 2013 Oct 28;288(42):30356-30364. Epub 2013 Aug 28.

From Haskins Laboratories and; the Department of Chemistry and Physical Sciences, Pace University, New York, New York 10038,. Electronic address:

Invasion of human intestinal epithelial cells (HCT-8) by Cryptosporidium parvum resulted in a rapid induction of host cell spermidine/spermine N(1)-acetyltransferase 1 (hSSAT-1) mRNA, causing a 4-fold increase in SSAT-1 enzyme activity after 24 h of infection. In contrast, host cell SSAT-2, spermine oxidase, and acetylpolyamine oxidase (hAPAO) remained unchanged during this period. Intracellular polyamine levels of C. parvum-infected human epithelial cells were determined, and it was found that spermidine remained unchanged and putrescine increased by 2.5-fold after 15 h and then decreased after 24 h, whereas spermine decreased by 3.9-fold after 15 h. Concomitant with these changes, N(1)-acetylspermine and N(1)-acetylspermidine both increased by 115- and 24-fold, respectively. Increased SSAT-1 has previously been shown to be involved in the endoplasmic reticulum (ER) stress response leading to apoptosis. Several stress response proteins were increased in HCT-8 cells infected with C. parvum, including calreticulin, a major calcium-binding chaperone in the ER; GRP78/BiP, a prosurvival ER chaperone; and Nrf2, a transcription factor that binds to antioxidant response elements, thus activating them. However, poly(ADP-ribose) polymerase, a protein involved in DNA repair and programmed cell death, was decreased. Cumulatively, these results suggest that the invasion of HCT-8 cells by C. parvum results in an ER stress response by the host cell that culminates in overexpression of host cell SSAT-1 and elevated N(1)-acetylpolyamines, which can be used by a parasite that lacks ornithine decarboxylase.
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http://dx.doi.org/10.1074/jbc.M113.459735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3798500PMC
October 2013

Histone 3.3 participates in a self-sustaining cascade of apoptosis that contributes to the progression of chronic obstructive pulmonary disease.

Am J Respir Crit Care Med 2013 Sep;188(6):673-83

1 Department of Biochemistry and.

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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http://dx.doi.org/10.1164/rccm.201302-0342OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826185PMC
September 2013
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