Medicine and Health Sciences Commons^™

Phi-1, An Endogenous Inhibitor Protein For Protein Phosphatase-1 And A Pan-Cancer Marker, Regulates Raf-1 Proteostasis, Jason Kirkbride, Garbo Nilsson, Jee In Kim, Kosuke Takeya, Yoshinori Tanaka, Hiroshi Tokumitsu, Futoshi Suizu, Masumi Eto

Kimmel Cancer Center Faculty Papers

Raf-1, a multifunctional kinase, regulates various cellular processes, including cell proliferation, apoptosis, and migration, by phosphorylating MAPK/ERK kinase and interacting with specific kinases. Cellular Raf-1 activity is intricately regulated through pathways involving the binding of regulatory proteins, direct phosphorylation, and the ubiquitin-proteasome axis. In this study, we demonstrate that PHI-1, an endogenous inhibitor of protein phosphatase-1 (PP1), plays a pivotal role in modulating Raf-1 proteostasis within cells. Knocking down endogenous PHI-1 in HEK293 cells using siRNA resulted in increased cell proliferation and reduced apoptosis. This heightened cell proliferation was accompanied by a 15-fold increase in ERK1/2 phosphorylation. Importantly, the observed …

Antibody Responses To Sars-Cov-2 After Infection Or Vaccination In Children And Young Adults With Inflammatory Bowel Disease., Joelynn Dailey, Lina Kozhaya, Mikail Dogan, Dena Hopkins, Blaine Lapin, Katherine Herbst, Michael Brimacombe, Kristen Grandonico, Fatih Karabacak, John Schreiber, Bruce Tsan-Liang Liang, Juan C Salazar, Derya Unutmaz, Jeffrey S Hyams

Faculty Research 2022

BACKGROUND: Characterization of neutralization antibodies to SARS-CoV-2 infection or vaccination in children and young adults with inflammatory bowel disease (IBD) receiving biologic therapies is crucial.

METHODS: We performed a prospective longitudinal cohort study evaluating SARS-CoV-2 spike protein receptor binding domain (S-RBD) IgG positivity along with consistent clinical symptoms in patients with IBD receiving infliximab or vedolizumab. Serum was also obtained following immunization with approved vaccines. The IgG antibody to the spike protein binding domain of SARS-CoV-2 was assayed with a fluorescent bead-based immunoassay that takes advantage of the high dynamic range of fluorescent molecules using flow cytometry. A sensitive and …

Glis1 Regulates Trabecular Meshwork Function And Intraocular Pressure And Is Associated With Glaucoma In Humans., K Saidas Nair, Chitrangda Srivastava, Robert V Brown, Swanand Koli, Hélène Choquet, Hong Soon Kang, Yien-Ming Kuo, Sara A Grimm, Caleb Sutherland, Alexandra Badea, G Allan Johnson, Yin Zhao, Jie Yin, Kyoko Okamoto, Graham Clark, Terete Borrás, Gulab Zode, Krishnakumar Kizhatil, Subhabrata Chakrabarti, Simon W M John, Eric Jorgenson, Anton M Jetten

Faculty Research 2021

Chronically elevated intraocular pressure (IOP) is the major risk factor of primary open-angle glaucoma, a leading cause of blindness. Dysfunction of the trabecular meshwork (TM), which controls the outflow of aqueous humor (AqH) from the anterior chamber, is the major cause of elevated IOP. Here, we demonstrate that mice deficient in the Krüppel-like zinc finger transcriptional factor GLI-similar-1 (GLIS1) develop chronically elevated IOP. Magnetic resonance imaging and histopathological analysis reveal that deficiency in GLIS1 expression induces progressive degeneration of the TM, leading to inefficient AqH drainage from the anterior chamber and elevated IOP. Transcriptome and cistrome analyses identified several glaucoma- …

Unraveling The Functional Role Of The Orphan Solute Carrier, Slc22a24 In The Transport Of Steroid Conjugates Through Metabolomic And Genome-Wide Association Studies, Sook Wah Yee, Adrian Stecula, Huan-Chieh Chien, Ling Zou, Elena V Feofanova, Marjolein Van Borselen, Kit Wun Kathy Cheung, Noha A Yousri, Karsten Suhre, Jason M Kinchen, Eric Boerwinkle, Roshanak Irannejad, Bing Yu, Kathleen M Giacomini

Journal Articles

Variation in steroid hormone levels has wide implications for health and disease. The genes encoding the proteins involved in steroid disposition represent key determinants of interindividual variation in steroid levels and ultimately, their effects. Beginning with metabolomic data from genome-wide association studies (GWAS), we observed that genetic variants in the orphan transporter, SLC22A24 were significantly associated with levels of androsterone glucuronide and etiocholanolone glucuronide (sentinel SNPs p-value

A Rational Approach For Creating Peptides Mimicking Antibody Binding, Sameer Sachdeva, Hyun Joo, Jerry Tsai, Bhaskara Jasti, Xiaoling Li

School of Pharmacy Faculty Articles

This study reports a novel method to design peptides that mimic antibody binding. Using the Knob-Socket model for protein-protein interaction, the interaction surface between Cetuximab and EGFR was mapped. EGFR binding peptides were designed based on geometry and the probability of the mapped knob-sockets pairs. Designed peptides were synthesized and then characterized for binding specificity, affinity, cytotoxicity of drug-peptide conjugate and inhibition of phosphorylation. In cell culture studies, designed peptides specifically bind and internalize to EGFR overexpressing cells with three to four-fold higher uptake compared to control cells that do not overexpress EGFR. The designed peptide, Pep11, bound to EGFR …

Proteomics Of Human Liver Membrane Transporters: A Focus On Fetuses And Newborn Infants., Bianca D. Van Groen, Evita Van De Steeg, Miriam G. Mooij, Marola M H Van Lipzig, Barbara A E De Koning, Robert M. Verdijk, Heleen M. Wortelboer, R Gaedigk, Chengpeng Bi, J Steven Leeder, Ron H N Van Schaik, Joost Van Rosmalen, Dick Tibboel, Wouter H. Vaes, Saskia N. De Wildt

Manuscripts, Articles, Book Chapters and Other Papers

BACKGROUND: Hepatic membrane transporters are involved in the transport of many endogenous and exogenous compounds, including drugs. We aimed to study the relation of age with absolute transporter protein expression in a cohort of 62 mainly fetus and newborn samples.

METHODS: Protein expressions of BCRP, BSEP, GLUT1, MCT1, MDR1, MRP1, MRP2, MRP3, NTCP, OCT1, OATP1B1, OATP1B3, OATP2B1 and ATP1A1 were quantified with LC-MS/MS in isolated crude membrane fractions of snap-frozen post-mortem fetal and pediatric, and surgical adult liver samples. mRNA expression was quantified using RNA sequencing, and genetic variants with TaqMan assays. We explored relationships between protein expression and age …

N-Glycosylation Regulates Pannexin 2 Localization But Is Not Required For Interacting With Pannexin 1., Rafael E Sanchez-Pupo, Danielle Johnston, Silvia Penuela

Anatomy and Cell Biology Publications

Pannexins (Panx1, 2, 3) are channel-forming glycoproteins expressed in mammalian tissues. We previously reported that N-glycosylation acts as a regulator of the localization and intermixing of Panx1 and Panx3, but its effects on Panx2 are currently unknown. Panx1 and Panx2 intermixing can regulate channel properties, and both pannexins have been implicated in neuronal cell death after ischemia. Our objectives were to validate the predicted N-glycosylation site of Panx2 and to study the effects of Panx2 glycosylation on localization and its capacity to interact with Panx1. We used site-directed mutagenesis, enzymatic de-glycosylation, cell-surface biotinylation, co-immunoprecipitation, and confocal microscopy. Our results showed …

Dimerization Of The Voltage-Sensing Phosphatase Controls Its Voltage-Sensing And Catalytic Activity., Vamseedhar Rayaprolu, Perrine Royal, Karen Stengel, Guillaume Sandoz, Susy C Kohout

Cooper Medical School of Rowan University Faculty Scholarship

Multimerization is a key characteristic of most voltage-sensing proteins. The main exception was thought to be the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP). In this study, we show that multimerization is also critical for Ci-VSP function. Using coimmunoprecipitation and single-molecule pull-down, we find that Ci-VSP stoichiometry is flexible. It exists as both monomers and dimers, with dimers favored at higher concentrations. We show strong dimerization via the voltage-sensing domain (VSD) and weak dimerization via the phosphatase domain. Using voltage-clamp fluorometry, we also find that VSDs cooperate to lower the voltage dependence of activation, thus favoring the activation of Ci-VSP. Finally, using …

Regulation Of Kv2.1 Channel Inactivation By Phosphatidylinositol 4,5-Bisphosphate., Mayra Delgado-Ramírez, José J De Jesús-Pérez, Iván A Aréchiga-Figueroa, Jorge Arreola, Scott K Adney, Carlos A. Villalba-Galea, Diomedes E Logothetis, Aldo A Rodríguez-Menchaca

School of Pharmacy Faculty Articles

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a membrane phospholipid that regulates the function of multiple ion channels, including some members of the voltage-gated potassium (Kv) channel superfamily. The PIP2 sensitivity of Kv channels is well established for all five members of the Kv7 family and for Kv1.2 channels; however, regulation of other Kv channels by PIP2 remains unclear. Here, we investigate the effects of PIP2 on Kv2.1 channels by applying exogenous PIP2 to the cytoplasmic face of excised membrane patches, activating muscarinic receptors (M1R), or depleting endogenous PIP2 using a rapamycin-translocated 5-phosphatase (FKBP-Inp54p). Exogenous PIP2 rescued Kv2.1 channels from rundown and partially …

Cib2 Interacts With Tmc1 And Tmc2 And Is Essential For Mechanotransduction In Auditory Hair Cells, Arnaud P. J. Giese, Yi-Quan Tang, Ghanshyam P. Sinha, Michael R. Bowl, Adam C. Goldring, Andrew Parker, Mary J. Freeman, Steve D. M. Brown, Saima Riazuddin, Robert Fettiplace, William R. Schafer, Gregory I. Frolenkov, Zubair M. Ahmed

Physiology Faculty Publications

Inner ear hair cells detect sound through deflection of stereocilia, the microvilli-like projections that are arranged in rows of graded heights. Calcium and integrin-binding protein 2 is essential for hearing and localizes to stereocilia, but its exact function is unknown. Here, we have characterized two mutant mouse lines, one lacking calcium and integrin-binding protein 2 and one carrying a human deafness-related Cib2 mutation, and show that both are deaf and exhibit no mechanotransduction in auditory hair cells, despite the presence of tip links that gate the mechanotransducer channels. In addition, mechanotransducing shorter row stereocilia overgrow in hair cell bundles of …

Carisbamate Blockade Of T-Type Voltage-Gated Calcium Channels, Do Young Kim, Fang-Xiong Zhang, Stan T. Nakanishi, Timothy Mettler, Ik-Hyun Cho, Younghee Ahn, Florian Hiess, Lina Chen, Patrick G. Sullivan, S. R. Wayne Chen, Gerald W. Zamponi, Jong M. Rho

Spinal Cord and Brain Injury Research Center Faculty Publications

Objectives

Carisbamate (CRS) is a novel monocarbamate compound that possesses antiseizure and neuroprotective properties. However, the mechanisms underlying these actions remain unclear. Here, we tested both direct and indirect effects of CRS on several cellular systems that regulate intracellular calcium concentration [Ca²⁺]_i.

Methods

We used a combination of cellular electrophysiologic techniques, as well as cell viability, Store Overload‐Induced Calcium Release (SOICR), and mitochondrial functional assays to determine whether CRS might affect [Ca²⁺]_i levels through actions on the endoplasmic reticulum (ER), mitochondria, and/or T‐type voltage‐gated Ca²⁺ channels.

Results

In CA3 pyramidal neurons, kainic …

Widespread Tau Seeding Activity At Early Braak Stages, Jennifer L. Furman, Jaime Vaquer-Alicea, Charles L. White, Nigel J. Cairns, Peter T. Nelson, Marc I. Diamond

Pathology and Laboratory Medicine Faculty Publications

Transcellular propagation of tau aggregates may underlie the progression of pathology in Alzheimer's disease (AD) and other tauopathies. Braak staging (B1, B2, B3) is based on phospho-tau accumulation within connected brain regions: entorhinal cortex (B1); hippocampus/limbic system (B2); and frontal and parietal lobes (B3). We previously developed a specific and sensitive assay that uses flow cytometry to quantify tissue seeding activity based on fluorescence resonance energy transfer (FRET) in cells that stably express tau reporter proteins. In a tauopathy mouse model, we have detected seeding activity far in advance of histopathological changes. It remains unknown whether individuals with AD also …

Identification Of Ecdysone Hormone Receptor Agonists As A Therapeutic Approach For Treating Filarial Infections, Amruta S. Mhashilkar, Sai L. Vankayala, Canhui Liu, Fiona Kearns, Priyanka Mehrotra, George Tzertzinis, Subba R. Palli, H. Lee Woodcock, Thomas R. Unnasch

Entomology Faculty Publications

Background A homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets.

Methodology/ Principal Findings Administration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at …

The Exceptionally High Reactivity Of Cys 621 Is Critical For Electrophilic Activation Of The Sensory Nerve Ion Channel Trpa1, Parmvir K. Bahia, Thomas A. Parks, Katherine R. Stanford, David A. Mitchell, Sameer Varma, Stanley M. Stevens Jr., Thomas E. Taylor-Clark

Molecular Biosciences Faculty Publications

Activation of the sensory nerve ion channel TRPA1 by electrophiles is the key mechanism that initiates nociceptive signaling, and leads to defensive reflexes and avoidance behaviors, during oxidative stress in mammals. TRPA1 is rapidly activated by subtoxic levels of electrophiles, but it is unclear how TRPA1 outcompetes cellular antioxidants that protect cytosolic proteins from electrophiles. Here, using physiologically relevant exposures, we demonstrate that electrophiles react with cysteine residues on mammalian TRPA1 at rates that exceed the reactivity of typical cysteines by 6,000-fold and that also exceed the reactivity of antioxidant enzymes. We show that TRPA1 possesses a complex reactive cysteine …

Steroid Binding To Autotaxin Links Bile Salts And Lysophosphatidic Acid Signalling, Willem-Jan Keune, Jens Hausmann, Ruth Bolier, Dagmar Tolenaars, Andreas Kremer, Tatjana Heidebrecht, Robbie P. Joosten, Manjula Sunkara, Andrew J. Morris, Elisa Matas-Rico, Wouter H. Moolenaar, Ronald P. Oude Elferink, Anastassis Perrakis

Gill Heart & Vascular Institute Faculty Publications

Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors …

Equine Arteritis Virus Uses Equine Cxcl16 As An Entry Receptor, Sanjay Sarkar, Lakshman Chelvarajan, Yun Young Go, Frank Cook, Sergey Artiushin, Shankar Mondal, Kelsi Anderson, John E. Eberth, Peter J. Timoney, Theodore S. Kalbfleisch, Ernest F. Bailey, Udeni B. R. Balasuriya

Veterinary Science Faculty Publications

Previous studies in our laboratory have identified equine CXCL16 (EqCXCL16) to be a candidate molecule and possible cell entry receptor for equine arteritis virus (EAV). In horses, the CXCL16 gene is located on equine chromosome 11 (ECA11) and encodes a glycosylated, type I transmembrane protein with 247 amino acids. Stable transfection of HEK-293T cells with plasmid DNA carrying EqCXCL16 (HEK-EqCXCL16 cells) increased the proportion of the cell population permissive to EAV infection from < 3% to almost 100%. The increase in permissiveness was blocked either by transfection of HEK-EqCXCL16 cells with small interfering RNAs (siRNAs) directed against EqCXCL16 or by pretreatment with guinea pig polyclonal antibody against EqCXCL16 protein (Gp anti-EqCXCL16 pAb). Furthermore, using a virus overlay protein-binding assay (VOPBA) in combination with far-Western blotting, gradient-purified EAV particles were shown to bind directly to the EqCXCL16 protein in vitro. The binding of biotinylated virulent EAV strain Bucyrus at 4°C was significantly higher in HEK-EqCXCL16 cells than nontransfected HEK-293T cells. Finally, the results demonstrated …

Translation Initiation Complex Eif4f Is A Therapeutic Target For Dual Mtor Kinase Inhibitors In Non-Hodgkin Lymphoma., Christos Demosthenous, Jing Jing Han, Mary J Stenson, Matthew J Maurer, Linda E Wellik, Brian Link, Kristen Hege, Ahmet Dogan, Eduardo Sotomayor, Thomas Witzig, Mamta Gupta

Medicine Faculty Publications

Deregulated mRNA translation has been implicated in disease development and in part is controlled by a eukaryotic initiation complex eIF4F (composed of eIF4E, eIF4G and eIF4A). We demonstrate here that the cap bound fraction from lymphoma cells was enriched with eIF4G and eIF4E indicating that lymphoma cells exist in an activated translational state. Moreover, 77% (110/142) of diffuse large B cell lymphoma tumors expressed eIF4E and this was associated with an inferior event free survival. Over-expression of wild-type eIF4E (eIF4E(WT)) but not cap-mutant eIF4E (eIF4E(cap mutant)) increased the activation of the eIF4F complex. Treatment with the active-site dual mTOR inhibitor …

Zhx2 Enhances The Cytotoxicity Of Chemotherapeutic Drugs In Liver Tumor Cells By Repressing Mdr1 Via Interfering With Nf-Ya, Hongxin Ma, Xuetian Yue, Lifen Gao, Xiaohong Liang, Wenjiang Yan, Zhenyu Zhang, Haixia Shan, Hualin Zhang, Brett T. Spear, Chunhong Ma

Microbiology, Immunology, and Molecular Genetics Faculty Publications

We previously reported the tumor suppressor function of Zinc-fingers and homeoboxes 2 (ZHX2) in hepatocellular carcinoma (HCC). Other studies indicate the association of increased ZHX2 expression with improved response to high dose chemotherapy in multiple myeloma. Here, we aim to test whether increased ZHX2 levels in HCC cells repress multidrug resistance 1(MDR1) expression resulting in increased sensitivity to chemotherapeutic drugs. We showed evidence that increased ZHX2 levels correlated with reduced MDR1 expression and enhanced the cytotoxicity of CDDP and ADM in different HCC cell lines. Consistently, elevated ZHX2 significantly reduced ADM efflux in HepG2 cells and greatly increased the CDDP-mediated …

The Rak/Frk Tyrosine Kinase Associates With And Internalizes The Epidermal Growth Factor Receptor, Ling Jin, Rolf J. Craven

Pharmacology and Nutritional Sciences Faculty Publications

Src is the founding member of a diverse family of intracellular tyrosine kinases, and Src has a key role in promoting cancer growth, in part, through its association with receptor tyrosine kinases. However, some Src-related proteins have widely divergent physiological roles, and these proteins include the Rak/Frk tyrosine kinase (Frk stands for Fyn-related kinase), which inhibits cancer cell growth and suppresses tumorigenesis. Rak/Frk phosphorylates and stabilizes the Pten tumor suppressor, protecting it from degradation, and Rak/Frk associates with the retinoblastoma (Rb) tumor suppressor. However, the role of Rak/Frk in receptor-mediated signaling is largely unknown. Here, we demonstrate that Rak/Frk associates …

Hiv-1 Tat Triggers Nuclear Localization Of Zo-1 Via Rho Signaling And Camp Response Element-Binding Protein Activation, Yu Zhong, Bei Zhang, Sung Yong Eum, Michal Toborek

Neurosurgery Faculty Publications

The human immunodeficiency virus (HIV)-specific protein trans-activator of transcription (Tat) can contribute to the dysfunction of brain endothelial cells and HIV trafficking into the brain by disrupting tight junction (TJ) integrity at the blood–brain barrier (BBB) level. Specific TJ proteins, such as zonula occludens (ZO) proteins, localize not only at the cell–cell borders but are also present in the nuclei. The objective of the present study was to evaluate the mechanisms and significance of Tat-induced nuclear localization of ZO-1. Treatment of a brain endothelial cell line (hCMEC/D3 cells) with Tat resulted in a decrease in total levels of ZO-1 but …

Targeted Over-Expression Of Glutamate Transporter 1 (Glt-1) Reduces Ischemic Brain Injury In A Rat Model Of Stroke, Brandon K. Harvey, Mikko Airavaara, Jason Michael Hinzman, Emily M. Wires, Matthew J. Chiocco, Douglas B. Howard, Hui Shen, Greg A. Gerhardt, Barry J. Hoffer, Yun Wang

Neuroscience Faculty Publications

Following the onset of an ischemic brain injury, the excitatory neurotransmitter glutamate is released. The excitotoxic effects of glutamate are a major contributor to the pathogenesis of a stroke. The aim of this study was to examine if overexpression of a glutamate transporter (GLT-1) reduces ischemic brain injury in a rat model of stroke. We generated an adeno-associated viral (AAV) vector expressing the rat GLT-1 cDNA (AAV-GLT1). Functional expression of AAV-GLT1 was confirmed by increased glutamate clearance rate in non-stroke rat brain as measured by in vivo amperometry. AAV-GLT1 was injected into future cortical region of infarction 3 weeks prior …