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Natasha Zachara, Ph.D.

Natasha Zachara, Ph.D.

Research in the Zachara Lab

The modification of nuclear, cytoplasmic, and mitochondrial proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) is an essential post-translational modification common in metazoans. O-GlcNAc is cycled on and off proteins in response to environmental and physiological stimuli impacting protein function, which, in turn, tunes pathways that include transcription, translation, proteostasis, signal transduction, and metabolism. In 2004 we demonstrated that O-GlcNAcylation of intracellular proteins was a novel regulator of cell survival and a modulator of the cellular stress response. In response to diverse forms of cellular stress, the O-GlcNAc modification is increased on myriad proteins. Elevation of O-GlcNAc levels prior to or after the induction of injury promotes cell survival in models of heat stress, hypoxia, oxidative stress, ischemia reperfusion injury, and trauma hemorrhage. Together, these data suggest that modification of proteins by O-GlcNAc in an integral component of the cellular stress response.

Our current work aims to define the mechanisms by which O-GlcNAc regulates cell survival in diverse models, understand how these mechanisms are dysregulated in disease, and to harness this information with the goal of improving patient outcomes. To identify key O-GlcNAcylated proteins that regulate cell survival decisions, we have used quantitative proteomics to define the O-GlcNAcome of cells subjected to heat or oxidative stress (Zachara et al., 2011; Lee et al., 2016). These and other studies showed that O-GlcNAc inhibits GSK3β leading to enhanced heat shock protein expression (Kazemi et al., 2010) and that O-GlcNAc regulates DNA damage response pathways (Zhong et al., 2015). In complimentary work, we are focused on understanding the mechanisms that regulate the enzymes and metabolites essential for O-GlcNAc-cycling (Narayanan et al., 2023). This work encompasses changes in physiology (sex), signaling (injury), and disease (mutation). To this end, we have characterized tools (Groves and Zachara, 2017, Narayanan, Zahra et al., 2023) and streamlined approaches that enable quantitative assessment of enzymatic activity and enzyme-protein interactors (Groves et al., 2017; Martinez et al., 2021).

Biography

Natasha Zachara Ph.D. (Citations: 6442; H-index: 37; i10-index: 54)

Dr. Natasha Zachara is an Associate Professor of Biological Chemistry and Oncology at the Johns Hopkins School of Medicine. Dr. Zachara received her undergraduate degree in biotechnology (with honors) from Macquarie University in Sydney, Australia. Her dissertation, completed at Macquarie University, focused on developing new technologies to map and quantify site-specific changes in protein glycosylation. She completed postdoctoral studies in glycobiology at the Johns Hopkins University School of Medicine. Dr. Zachara joined the Johns Hopkins faculty in 2007. Currently, she is the Director of the K12 training program “Immersive Training in the Glycosciences”, Chair of the “Glycan Advisory Committee”, a Director of the “Society for Glycobiology”, and an Associate Editor of the Biochemical Journal.

Current Zachara Lab Members

  • Fiddia Zahra, Graduate Student
  • Akanksha Aggarwal, Graduate Student
  • Megan Craven, Undergraduate Student
  • Sandra Fahmy, Undergraduate Student

Recent and Key Citations:

  • Narayanan B, Sinha P, Henry R, Reeves RA, Paolocci N, Kohr MJ, Zachara NE. Cardioprotective O-GlcNAc-Signaling is Elevated in Murine Female Hearts via Enhanced O-GlcNAc Transferase Activity. J Biol Chem. 2023 Nov 8:105447. PMID: 37949223.
  • Narayanan B, Zahra F, Reeves RA, Aggarwal A, O’Meally RN, Henry R, Craven M, Jacobson A, Cole RN, Kohr M, Umapathi P, Zachara NE. Differential Detection of O-GlcNAcylated proteins in the heart using antibodies. Anal Biochem. 2023 Jul 26:115262. doi: 10.1016/j.ab.2023.115262. Epub ahead of print. PMID: 37507081.
  • Martinez M, Renuse S, Kreimer S, O’Meally R, Natov P, Madugundu AK, Nirujogi RS, Tahir R, Cole R, Pandey A, Zachara NE. Quantitative Proteomics Reveals that the OGT Interactome Is Remodeled in Response to Oxidative Stress. Mol Cell Proteomics. 2021 Mar 12;20:100069. PMID: 33716169; PMCID: PMC8079276.
  • Mesubi OO, Rokita AG, Abrol N, Wu Y, Chen B, Wang Q, Granger JM, Tucker-Bartley A, Luczak ED, Murphy KR, Umapathi P, Banerjee PS, Boronina TN, Cole RN, Maier LS, Wehrens XH, Pomerantz JL, Song LS, Ahima RS, Hart GW, Zachara NE, Anderson ME. Oxidized CaMKII and O-GlcNAcylation cause increased atrial fibrillation in diabetic mice by distinct mechanisms. J Clin Invest. 2021 Jan 19;131(2):e95747. PMID: 33151911; PMCID: PMC7810480.
  • Umapathi P, Mesubi OO, Banerjee PS, Abrol N, Wang Q, Luczak ED, Wu Y, Granger JM, Wei AC, Reyes Gaido OE, Florea L, Talbot CC Jr, Hart GW, Zachara NE, Anderson ME. Excessive O-GlcNAcylation Causes Heart Failure and Sudden Death. Circulation. 2021 Apr 27;143(17):1687-1703. PMID: 33593071; PMCID: PMC8085112.
  • Groves JA, Maduka AO, O’Meally RN, Cole RN, Zachara NE. Fatty acid synthase inhibits the O-GlcNAcase during oxidative stress. J Biol Chem. 2017 Apr 21;292(16):6493-6511. PMID: 28232487; PMCID: PMC5399103.
  • Zhong J, Martinez M, Sengupta S, Lee A, Wu X, Chaerkady R, Chatterjee A, O’Meally RN, Cole RN, Pandey A, Zachara NE. Quantitative phosphoproteomics reveals crosstalk between phosphorylation and O-GlcNAc in the DNA damage response pathway. Proteomics. 2015 Jan;15(2-3):591-607. PMID: 25263469; PMCID: PMC4564869.
  • Lee A, Miller D, Henry R, Paruchuri VD, O’Meally RN, Boronina T, Cole RN, Zachara NE. Combined Antibody/Lectin Enrichment Identifies Extensive Changes in the O-GlcNAc Sub-proteome upon Oxidative Stress. J Proteome Res. 2016 Dec 2;15(12):4318-4336. PMID: 27669760.
  • Zachara NE, Molina H, Wong KY, Pandey A, Hart GW. The dynamic stress-induced “O-GlcNAc-ome” highlights functions for O-GlcNAc in regulating DNA damage/repair and other cellular pathways. Amino Acids. 2011 Mar;40(3):793-808. PMID: 20676906; PMCID: PMC3329784.
  • Kazemi Z, Chang H, Haserodt S, McKen C, Zachara NE. O-linked beta-N-acetylglucosamine (O-GlcNAc) regulates stress-induced heat shock protein expression in a GSK-3beta-dependent manner. J Biol Chem. 2010 Dec 10;285(50):39096-107. PMID: 20926391; PMCID: PMC2998145.
  • Zachara NE, O’Donnell N, Cheung WD, Mercer JJ, Marth JD, Hart GW. Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress. A survival response of mammalian cells. J Biol Chem. 2004 Jul 16;279(29):30133-42. PMID: 15138254.

A complete list of publications: https://www.ncbi.nlm.nih.gov/myncbi/natasha.zachara.1/bibliography/public/.

Joel L. Pomerantz, Ph.D.

Joel L. Pomerantz, Ph.D.

Research Overview

My laboratory is interested in the molecular mechanisms by which cells interpret signals from their environment that instruct them to proliferate, differentiate, or die by apoptosis. This process is of fundamental importance in the development and function of the immune system. The dysregulation of signal transduction underlies many diseases of the immune system including immunodeficiencies, autoimmunity, and cancers derived from immune cells. A particular focus of the lab is the regulation of NF-κB, a pleiotropic transcription factor that is required for normal innate and adaptive immunity and which is inappropriately activated in several types of human cancer. We have been studying how NF-κB is activated in B and T lymphocytes in response to antigen recognition by the T cell receptor (TCR) and B cell receptor (BCR) complexes. Recently we have characterized the molecular mechanisms by which a multi-domain adapter protein, CARD11, functions in TCR signaling to NF-κB. In response to antigen receptor engagement, CARD11 undergoes a transition from an inactive to an active protein scaffold, and recruits a cadre of signaling cofactors into a complex in a signal-responsive manner. Current research is aimed at understanding how the multiple domains of CARD11 function together to translate activating upstream signals from the antigen receptor into the coordinated signaling activity of associated cofactors. CARD11 has also been directly implicated in the dysregulated signaling to NF-κB that is a signature feature of a subtype of Diffuse Large B cell Lymphoma (DLBCL). This subtype of DLBCL requires constitutive NF-κB activation for oncogenic proliferation, and the knockdown of CARD11 in this lymphoma leads to apoptosis. Interestingly, several oncogenic mutations in CARD11 have been identified in human DLBCL. We are currently studying how the oncogenic CARD11 mutations result in hyperactive signaling to NF-κB. We are hopeful that a mechanistic understanding of these mutants might translate into the development of novel cancer therapeutics. In addition to the antigen receptor signaling pathway, we are also studying the regulation of NF-κB in other arms of the innate and adaptive immune system through the isolation and characterization of novel regulators of NF-κB activity. We have developed several novel expression-cloning approaches for identifying novel signaling molecules that either activate or inhibit NF-κB. Several novel signaling regulators are under current study. Other current projects include the study of novel regulators of the NFAT transcription factor, a key player in T cell activation and tolerance. It is our hope that the study of these signaling molecules will expand our understanding of how inflammatory and immune responses are controlled and they are dysregulated in human disease.

Cellular Stress and Cell Signaling | Genetics, Genomics and Gene Regulation | Immunology and Infectious Diseases

 

Lab Website

Pomerantz Lab – Lab Website

 

Selected Publications

Mollie K. Meffert, M.D., Ph.D.

Mollie K. Meffert, M.D., Ph.D.

Background

Dr. Mollie Meffert is an associate professor of biological chemistry and neuroscience at the Johns Hopkins University School of Medicine. Her research focuses on the regulation of neuronal gene expression in health and disease. Dr. Meffert currently serves as the Vice Director of the Department of Biological Chemistry.

Research Description

The Meffert lab investigates mechanisms underlying enduring change in mammalian nervous system function in health and disease. We are interested in how cells in the nervous system make decisions to turn genes on or off, and how those decisions are remembered in processes such as development or plasticity, and in injury or disease. The goal of the Meffert lab is to gain a mechanistic understanding of how selective gene programs are recruited and maintained to alter synaptic, neuronal, and cognitive function. Rather than focusing on single genes, we investigate the upstream processes that allow coordinate regulation of many genes to achieve biological impact. Cell-specific and subcellularly localized posttranscriptional control by RNA-binding proteins and noncoding RNAs is an ongoing focus.

Our laboratory elucidated a post-transcriptional mechanism capable of organizing pro-growth gene programs in which activity-dependent regulation of microRNA (miRNA) production governs the selection of gene targets for protein synthesis. An RNA-binding protein, Lin28, is one activity-responsive factor that promotes pro-growth protein synthesis by downregulating only select miRNAs (e.g. the family of let-7 ‘growth-suppressor’ miRNAs), which repress pro-growth genes. In neurons, pro-growth mRNA targets of the let-7 miRNAs include mRNA for proteins involved in excitatory synaptic function, as well as growth, metabolism, and repair. In recent work, we develop discovery-based sequencing strategies to reveal in vivo small RNA targets through the production of small RNA:target chimeric molecules.

Dr. Meffert’s work has been recognized with awards including the: March of Dimes research scholar, Simons Foundation Autism Research Initiative award, PLU Rho Award, Alfred P. Sloan Research Fellow Award, Hamilton Smith Award for Innovative Science, and The Sontag Foundation Distinguished Scientist and Distinguished Alumni Awards.
Dr. Meffert and her laboratory are the current recipients of the Eric C. Aker Award Endowment through the Braude Foundation.

Expertise

Dr. Meffert received her undergraduate degree (BS) from Stanford University. She earned her MD/PhD in neuroscience from Stanford University School of Medicine and completed a postdoctoral fellowship at California Institute of Technology with Dr. David Baltimore.

Areas of research expertise in Dr. Meffert’s laboratory include molecular neuroscience, RNA biology, and gene expression. Her laboratory uses tools of molecular diagnostics, biochemistry, computational biology, quantitative imaging, and mouse and human genetic models of disease

Lab Members

Xinbei Li (BC graduate student), Bonita Powell (BCMB graduate student), Emily Eiss (BCMB graduate student), Ariella Kornfeld (BCMB graduate student), Sydney Pettit (BCMB graduate student), Preksha Jerajani (research technician), Anselmo Rivera (Hopkins undergraduate)

Selected Publications

  • Xinbei Li, William T. Mills IV, Daniel S. Jin, and Mollie K.Meffert. (2024), Genome-wide and cell-type-selective profiling of in vivo small noncoding RNA:target RNA interactions by chimeric RNA sequencing. Cell Reports Methods, 4, 100836.
  • Megha Subramanian, William T. Mills IV, Manish D Paranjpe, Uche Onuchukwu, Manasi Inamdar, Amanda R. Maytin, Xinbei Li, Joel L. Pomerantz, and Mollie K.Meffert. (2023), Growth suppressor microRNAs mediate synaptic overgrowth and behavioral deficits in Fragile X mental retardation protein deficiency. iScience, 27 (1) 108676.
  • William T. Mills IV, Sreenivas Eadara, Andrew E. Jaffe, and Mollie K. Meffert. (2022), SCRAP: a bioinformatic pipeline for the analysis of small chimeric RNA-seq data. RNA 29 (1); 1-17.
  • Alexandra M Amen, Claudia R. Ruiz, Jay Shi, Megha Subramanian, Daniel Pham, and Mollie K. Meffert, (2017) A rapid induction mechanism for Lin28a in trophic responses. Molecular Cell, 65 (3); 490 – 503.
  • Erica C. Dresselhaus, Matthew C. Boersma, and Mollie K. Meffert, (2018), Targeting of NF-kB to dendritic spines is required for synaptic signaling and spine development. J.Neurosci., 8(17); 4093-4103. PMID 29555853.
  • Laurel M. Oldach, Kirill Gorshkov,William T. Mills, Jin Zhang*, and Mollie Meffert* (2018), A biosensor for MAPK-dependent Lin28 signaling. Molecular Biology of the Cell, 29(10), 1157-1167. PMID29540527.
  • Yu-Wen A. Huang*, Claudia R. Ruiz*, E.C.H. Eyler*, Kathie Lin, and Mollie K. Meffert. “Dual regulation of miRNA biogenesis generates target specificity in neurotrophin-induced protein synthesis.” Cell, 148(5); 933-946. 2012.
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