Life Sciences Commons^™

Engineered Nls-Chimera Downregulates Expression Of Aggregation-Prone Endogenous Fus, Miyuki Hayashi, Amandeep Girdhar, Ying-Hui Ko, Kevin Kim, Jacquelyn Depierro, Joseph R. Buchler, Nikhita Arunprakash, Aditya Bajaj, Gino Cingolani, Lin Guo

Department of Biochemistry and Molecular Biology Faculty Papers

Importin β-superfamily nuclear import receptors (NIRs) mitigate mislocalization and aggregation of RNA-binding proteins (RBPs), like FUS and TDP-43, which are implicated in neurodegenerative diseases. NIRs potently disaggregate RBPs by recognizing their nuclear localization signal (NLS). However, disease-causing mutations in NLS compromise NIR binding and activity. Here, we define features that characterize the anti-aggregation activity of NIR and NLS. We find that high binding affinity between NIR and NLS, and optimal NLS location relative to the aggregating domain plays a role in determining NIR disaggregation activity. A designed FUS chimera (FUS_IBB), carrying the importin β binding (IBB) domain, is …

Methane Fluxes In Tidal Marshes Of The Conterminous United States, Ariane Arias-Ortiz, Jaxine Wolfe, Scott D. Bridgham, Sara Knox, Gavin Mcnicol, Brian A. Needelman, Julie Shahan, Ellen J. Stuart-Haëntjens, Lisamarie Windham-Myers, Patty Y. Oikawa, Dennis D. Baldocchi, Joshua S. Caplan, Margaret Capooci, Kenneth M. Czapla, R. Kyle Derby, Heida L. Diefenderfer, Inke Forbrich, Gina Groseclose, Jason K. Keller, Cheryl Kelley, Amir E. Keshta, Helena S. Kleiner, Ken W. Krauss, Robert R. Lane, Sarah Mack, Serena Moseman-Valtierra, Thomas J. Mozdzer, Peter Mueller, Scott C. Neubauer, Genevieve Noyce, Katrina V. R. Schäfer, Rebecca Sanders-Demott, Charles A. Schutte, Rodrigo Vargas, Nathaniel B. Weston, Benjamin Wilson, J. Patrick Megonigal, James R. Homquist

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Methane (CH4) is a potent greenhouse gas (GHG) with atmospheric concentrations that have nearly tripled since pre-industrial times. Wetlands account for a large share of global CH4 emissions, yet the magnitude and factors controlling CH4 fluxes in tidal wetlands remain uncertain. We synthesized CH4 flux data from 100 chamber and 9 eddy covariance (EC) sites across tidal marshes in the conterminous United States to assess controlling factors and improve predictions of CH4 emissions. This effort included creating an open-source database of chamber-based GHG fluxes (https://doi.org/10.25573/serc.14227085). Annual fluxes across chamber and EC sites averaged 26 ± 53 g CH4 …

Structural Basis For Substrate Binding And Selection By Human Mitochondrial Rna Polymerase, Karl Herbine, Ashok Nayak, Dmitry Temiakov

Department of Biochemistry and Molecular Biology Faculty Papers

The mechanism by which RNAP selects cognate substrates and discriminates between deoxy and ribonucleotides is of fundamental importance to the fidelity of transcription. Here, we present cryo-EM structures of human mitochondrial transcription elongation complexes that reveal substrate ATP bound in Entry and Insertion Sites. In the Entry Site, the substrate binds along the O helix of the fingers domain of mtRNAP but does not interact with the templating DNA base. Interactions between RNAP and the triphosphate moiety of the NTP in the Entry Site ensure discrimination against nucleosides and their diphosphate and monophosphate derivatives but not against non-cognate rNTPs and …

Stem-Loop And Circle-Loop Tads Generated By Directional Pairing Of Boundary Elements Have Distinct Physical And Regulatory Properties, Wenfan Ke, Miki Fujioka, Paul Schedl, James Jaynes

Department of Biochemistry and Molecular Biology Faculty Papers

The chromosomes in multicellular eukaryotes are organized into a series of topologically independent loops called TADs. In flies, TADs are formed by physical interactions between neighboring boundaries. Fly boundaries exhibit distinct partner preferences, and pairing interactions between boundaries are typically orientation-dependent. Pairing can be head-to-tail or head-to-head. The former generates a stem-loop TAD, while the latter gives a circle-loop TAD. The TAD that encompasses the Drosophila even skipped (eve) gene is formed by the head-to-tail pairing of the nhomie and homie boundaries. To explore the relationship between loop topology and the physical and regulatory landscape, we flanked the nhomie boundary …

G12/13 Signaling In Asthma, Elizabeth L. Mcduffie, Reynold A Panettieri, Charles P. Scott

Department of Biochemistry and Molecular Biology Faculty Papers

Shortening of airway smooth muscle and bronchoconstriction are pathognomonic for asthma. Airway shortening occurs through calcium-dependent activation of myosin light chain kinase, and RhoA-dependent calcium sensitization, which inhibits myosin light chain phosphatase. The mechanism through which pro-contractile stimuli activate calcium sensitization is poorly understood. Our review of the literature suggests that pro-contractile G protein coupled receptors likely signal through G12/13 to activate RhoA and mediate calcium sensitization. This hypothesis is consistent with the effects of pro-contractile agonists on RhoA and Rho kinase activation, actin polymerization and myosin light chain phosphorylation. Recognizing the likely role of G12/13 signaling in the pathophysiology …

Developing A Comprehensive Genome-Scale Metabolic Model For The Arabidopsis Thaliana Root System, Lohani Esterhuizen

Department of Chemical and Biomolecular Engineering: Dissertations, Theses, and Student Research

Arabidopsis thaliana (Arabidopsis) is the most well-established model plant to date. Being the first plant to have its genome mapped, studies on Arabidopsis have provided insurmountable insights into the physiological and biochemical nature of plants. Methods that allow us to computationally study the metabolism of organisms include the use of genome-scale metabolic models (GEMs). Despite its popularity, no GEM currently maps the metabolic activity in the root system of Arabidopsis, which is the first organ to face and respond to stress conditions in the soil. This work aims to develop and implement a comprehensive GEM of the Arabidopsis root system …

Synergistic Effects Of Novel Penicillin-Binding Protein 1a Amino Acid Substitutions Contribute To High-Level Amoxicillin Resistance Of Helicobacter Pylori, Alain Cimuanga-Mukanya, Evariste Tshibangu-Kabamba, Patrick De Jesus Ngoma Kisoko, Fabien Mbaya Tshibangu, Antoine Tshimpi Wola, Pascal Tshiamala Kashala, Dieudonné Mumba Ngoyi, Steve Ahuka-Mundeke, Gunturu Revathi, Ghislain Disashi-Tumba

Pathology, East Africa

The growing resistance to amoxicillin (AMX)—one of the main antibiotics used in Helicobacter pylori eradication therapy—is an increasing health concern. Several mutations of penicillin-binding protein 1A (PBP1A) are suspected of causing AMX resistance; however, only a limited set of these mutations have been experimentally explored. This study aimed to investigate four PBP1A mutations (i.e., T558S, N562H, T593A, and G595S) carried by strain KIN76, a high-level AMX-resistant clinical H. pylori isolate with an AMX minimal inhibition concentration (MIC) of 2 µg/mL. We transformed a recipient strain 26695 with the DNA containing one to four mutation allele combinations of the pbp1 gene …

Detection Of Cryptosporidium Spp. In Surface Water And Tissues Of Bivalves In Waterways Of Coney Island, New York (Usa), Manasvi Patel

Biochemistry and Molecular Biology

Combined sewer overflows (CSOs) are major sources of pollutants such as microbial pathogens, total suspended solids (TSS), oxygen depleting substances, toxicants, and nutrients. Untreated wastewater contains parasites like Giardia, Cryptosporidium and Entamoeba which impact human health. Cryptosporidium is the leading cause of diarrheal diseases which spreads through contaminated water supplies. Our research on Coney Island in Brooklyn investigated the amount of Cryptosporidium oocysts found in the water and bivalves/shellfish tissue and to supports a parallel study examining both people's use of waterways around Coney Island (for religious ceremonies, fishing, foraging, swimming, etc.) and their perceptions of how polluted the …

Role Of The Malate Dehydrogenase-Citrate Synthase Metabolon In Metabolic Flux Regulation, Joy John Omini

Dissertations and Doctoral Documents from University of Nebraska-Lincoln, 2023–

The TCA cycle enzymes- malate dehydrogenase (MDH) and citrate synthase (CS)- interact to transfer oxaloacetate from the active site of MDH to the active site of CS, through an electropositive channel. The MDH-CS metabolon is highly conserved across different domains of life, and it has been associated with many advantages. Some of these advantages include protecting oxaloacetate from competing enzymes like aspartate aminotransferase, allowing the thermodynamically unfavorable MDH forward reaction to occur and increasing the local concentration of oxaloacetate within the active site of CS. These advantages and conservativeness of the MDH-CS metabolon indicate that it is highly important to …

Mutant Androgen Receptor Induces Neurite Loss And Senescence Independently Of Are Binding In A Neuronal Model Of Sbma, Jordyn Karliner, Y Liu, Diane Merry

Department of Biochemistry and Molecular Biology Faculty Papers

Spinal and bulbar muscular atrophy (SBMA) is a slowly progressing neuromuscular disease caused by a polyglutamine (polyQ)-encoding CAG trinucleotide repeat expansion in the androgen receptor (AR) gene, leading to AR aggregation, lower motor neuron death, and muscle atrophy. AR is a ligand-activated transcription factor that regulates neuronal architecture and promotes axon regeneration; however, whether AR transcriptional functions contribute to disease pathogenesis is not fully understood. Using a differentiated PC12 cell model of SBMA, we identified dysfunction of polyQ-expanded AR in its regulation of neurite growth and maintenance. Specifically, we found that in the presence of androgens, polyQ-expanded AR inhibited neurite …

Structural Analysis Of Dj-1 Glyoxalase Activity By Mix-And-Inject Serial Synchrotron Crystallography, Coleman Dolamore

Department of Biochemistry: Dissertations, Theses, and Student Research

Observing enzyme structures while they are catalyzing a reaction has been a major goal of both enzymology and structural biology for decades. With the advent of time-resolved serial X-ray crystallography using X-ray free electron lasers (XFELs) and synchrotron sources, enzyme reactions can be monitored in real time in crystallo. Here, we use two different approaches to study related enzymes involved in methylglyoxal detoxification. For human DJ-1, we used pink beam mix-and-inject serial crystallography (MISC) at the Advanced Photon Source (BioCARS 14-ID) to probe the controversial mechanism of DJ-1’s action on methylglyoxal by using serial Laue diffraction. The high flux …

Identifying The Relative Abundance Of Cell-Cell Signaling Peptides During Mammalian Hibernation And Anesthetic Administration By Label-Free Peptidomics Analysis, Somayeh Mousavi

Dissertations and Doctoral Documents from University of Nebraska-Lincoln, 2023–

Cell-cell signaling molecules known as neuropeptide and peptide hormones, distributed in both the central nervous system (CNS) and the peripheral nervous system (PNS), regulate a variety of physiological processes including body temperature, feeding, circadian rhythm, reproduction, energy homeostasis, and more. While a number of active neuropeptides structures with physiological functions have been discovered, advances in peptidomic mass spectrometry are enabling researchers to uncover novel, previously unknown, active neuropeptide structures with potential for bioactivities. Using liquid chromatography coupled with tandem mass spectrometry (MS/MS), it is possible to identify a wide range of neuropeptides in single experiments without preselecting specific peptides. Mass …

Adipose-Derived Mesenchymal Stem Cells And Their Derived Epidermal Progenitor Cells Conditioned Media Ameliorate Skin Aging In Rats, Mohamed M. Kamal, Omar Ibrahim Badr, Tasnim Mahmoud

Pharmacy

BACKGROUND: Skin alterations are among the most prominent signs of aging, and they arise from both intrinsic and extrinsic factors that interact and mutually influence one another. The use of D-galactose as an aging model in animals has been widely employed in anti-aging research. Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) are particularly promising for skin anti-aging therapy due to their capacity for effective re-epithelization and secretion of various growth factors essential for skin regeneration. Accordingly, we aimed to examine the potential utility of Ad-MSCs as a therapy for skin anti-aging. METHODS: In this study, we isolated and characterized adipose-derived mesenchymal …

Structural And Biophysical Insights Into Targeting Of Claudin-4 By A Synthetic Antibody Fragment, Satchal K. Erramilli, Pawel K. Dominik, Chinemerem P. Ogbu, Anthony A. Kossiakoff, Alex J. J.

Department of Biochemistry: Faculty Publications

Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties—these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes …

Perspectives On Computational Modeling Of Biological Systems And The Significance Of The Sysmod Community, Bhanwar Lal Puniya, Meghna Verma, Chiara Damiani, Shaimaa Bakr, Andreas Dräger

Department of Biochemistry: Faculty Publications

Motivation: In recent years, applying computational modeling to systems biology has caused a substantial surge in both discovery and practical applications and a significant shift in our understanding of the complexity inherent in biological systems.

Results: In this perspective article, we briefly overview computational modeling in biology, highlighting recent advancements such as multiscale modeling due to the omics revolution, single-cell technology, and integration of artificial intelligence and machine learning approaches. We also discuss the primary challenges faced: integration, standardization, model complexity, scalability, and interdisciplinary collaboration. Lastly, we highlight the contribution made by the Computational Modeling of Biological Systems …

Frontotemporal Dementia-Like Disease Progression Elicited By Seeded Aggregation And Spread Of Fus, Sonia Vazquez-Sanchez, Britt Tilkin, Fatima Gasset-Rosa, Sitao Zhang, Diana Piol, Melissa Mcalonis-Downes, Jonathan Artates, Noe Govea-Perez, Yana Verresen, Lin Guo, Don Cleveland, James Shorter, Sandrine Da Cruz

Department of Biochemistry and Molecular Biology Faculty Papers

RNA binding proteins have emerged as central players in the mechanisms of many neurodegenerative diseases. In particular, a proteinopathy of fused in sarcoma (FUS) is present in some instances of familial Amyotrophic lateral sclerosis (ALS) and about 10% of sporadic Frontotemporal lobar degeneration (FTLD). Here we establish that focal injection of sonicated human FUS fibrils into brains of mice in which ALS-linked mutant or wild-type human FUS replaces endogenous mouse FUS is sufficient to induce focal cytoplasmic mislocalization and aggregation of mutant and wild-type FUS which with time spreads to distal regions of the brain. Human FUS fibril-induced FUS aggregation …

A Library Of 39 Plant Peroxidases: Plants From The Families Cucurbitaceae And Brassicaceae Show Promising Peroxidase And Peroxygenase Activity, Sally Hess

Senior Honors Theses

Peroxidases are enzymes that reduce reactive oxygen species (ROS) and oxidize organic substances in living cells. The textile industry, wastewater treatment plants, immunological laboratories, and diagnostic laboratories have extensively used horseradish peroxidase for their oxidating needs. This research aims to provide a tool for identifying potentially useful sources of peroxidases in addition to specifically pinpointing a novel peroxygenase in jalapeño seeds. By using two widely accepted peroxidase assays, the guaiacol assay and the fluorophenol assay, fruits of the genus Cucurbita and roots or leaves of the family Brassicaceae were seen to have high peroxidase activity. Additionally, novel peroxygenase activity was …

Descspim: An Affordable And Easy-To-Build Light-Sheet Microscope Optimized For Tissue Clearing Techniques, Kohei Otomo, Takaki Omura, Yuki Nozawa, Steven J. Edwards, Yukihiko Sato, Yuri Saito, Shigehiro Yagishita, Hitoshi Uchida, Yuki Watakabe, Kiyotada Naitou, Rin Yanai, Naruhiko Sahara, Satoshi Takagi, Ryohei Katayama, Yusuke Iwata, Toshiro Shiokawa, Yoku Hayakawa, Kensuke Otsuka, Haruko Watanabe-Takano, Yuka Haneda, Shigetomo Fukuhara, Miku Fujiwara, Takenobu Nii, Chikara Meno, Naoki Takeshita, Kenta Yashiro, Juan Marcelo Rosales Rocabado, Masaru Kaku, Tatsuya Yamada, Yumiko Oishi, Hiroyuki Koike, Yinglan Cheng, Keisuke Sekine, Jun-Ichiro Koga, Kaori Sugiyama, Kenichi Kimura, Fuyuki Karube, Hyeree Kim, Ichiro Manabe, Tomomi Nemoto, Kazuki Tainaka, Akinobu Hamada, Hjalmar Brismar, Etsuo A. Susaki

Department of Biochemistry: Faculty Publications

Despite widespread adoption of tissue clearing techniques in recent years, poor access to suitable light-sheet fluorescence microscopes remains a major obstacle for biomedical end-users. Here, we present descSPIM (desktopequipped SPIM for cleared specimens), a low-cost ($20,000–50,000), lowexpertise (one-day installation by a non-expert), yet practical do-it-yourself light-sheet microscope as a solution for this bottleneck. Even the most fundamental configuration of descSPIM enables multi-color imaging of whole mouse brains and a cancer cell line-derived xenograft tumor mass for the visualization of neurocircuitry, assessment of drug distribution, and pathological examination by false-colored hematoxylin and eosin staining in a threedimensional manner. Academically open-sourced (https://github.com/dbsbjuntendo/ …

Multifaceted Roles Of Meg3 In Cellular Senescence And Atherosclerosis, Xiao Cheng, Mohamed Sham Shihabudeen Haider Ali, Vijaya Bhaskar Baki, Matthew Moran, Huabo Su, Xinghui Sun

Department of Biochemistry: Faculty Publications

Background and aims: Long noncoding RNAs are involved in the pathogenesis of atherosclerosis. As long non-coding RNAs maternally expressed gene 3 (Meg3) prevents cellular senescence of hepatic vascular endothelium and obesity-induced insulin resistance, we decided to examine its role in cellular senescence and atherosclerosis.

Methods and Results: By analyzing our data and human and mouse data from the Gene Expression Omnibus database, we found that Meg3 expression was reduced in humans and mice with cardiovascular disease, indicating its potential role in atherosclerosis. In Ldlr^−/− mice fed a Western diet for 12 weeks, Meg3 silencing by chemically modified …

Chm116 Principles Of Chemistry Ii Lecture Slides, Leah S. Cohen, Probal Banerjee

Open Educational Resources

This file contains the lecture slides that are used in CHM116, Principles of Chemistry II.

Investigation Of How A Kinesin-1 Mutation Affects The Synaptic Localization Of The Periactive Zone Protein Apt-4, Ian Harden

Honors College

Dysfunctional or loss of synaptic transmission is a major contributing factor to many neurological and neurodevelopmental diseases, such as Alzheimer’s disease. To develop novel approaches to treating these diseases, understanding the structures and proteins related to synaptic development and function is essential. Within the synapse is the presynaptic site, containing the active and periactive zones, at which synaptic vesicles containing neurotransmitters are released. The active zone’s primary role is mediating neurotransmitter release via exocytosis, and the periactive zone sorts and recycles synaptic vesicles via endocytosis. However, how periactive zone proteins localize to the presynapse and how they interact with the …

A Review Of Rheb Activation Of Mtorc1 And The Great Mystery Of One Missing Gef, Jack Gregory

Senior Honors Theses

The mTORC1 pathway is involved in the regulation of cell growth and translation. The pathway has a complex web of activators and inhibitors to activate mTORC1. mTORC1 is regulated via a small GTPase called Rheb, which interacts directly with mTORC1. This GTPase and its GTPase activating protein (GAP), TSC1/2, have been widely studied to understand how the variety of regulators of mTORC1 interact with these proteins. Despite this, the guanine nucleotide exchange factor (GEF) of Rheb has yet to be identified. This review broadly analyzes Rheb and mTORC1, their structures, regulations, and interactions, and explores the mystery of the missing …

A Combination Of Generated Hydrogen Sulfide And Nitric Oxide Activity Has A Potentiated Protectant Effect Against Cisplatin Induced Nephrotoxicity, Faria Khurshid, Javeid Iqbal, Fiaz Ud Din Ahmad, Arslan Hussain Lodhi, Abdul Malik, Suhail Akhtar, Azmat Ali Khan, Marvi Imam Bux, Mohammed Younis

Biomedical Sciences Faculty Publications

Aim: Hydrogen sulfide and nitricoxide possess cytoprotective activity and in vivo, they are generated from exogenous sodium hydrosulfide and L-arginine respectively. Cisplatin is a major chemotherapeutic agent used to treat cancer and has a high incidence of nephrotoxicity as a side effect. The study aim was to explore the effects of NaHS and L-arginine or their combination on cisplatin induced nephrotoxicity in rats. Methods: Wistar Kyoto rats were given a single intraperitoneal dose of cisplatin (5 mg/kg) followed either by NaHS (56 μmol/kg, i. p.), L-arginine (1.25 g/L in drinking water) or their combination daily for 28-days. Post-mortem plasma, urine …

The Purification And Thermal Stability Of The Peroxidase Enzyme In Cucurbita Moschata, Garen Hamner

Senior Honors Theses

Peroxidases are enzymes that catalyze the reduction of hydrogen peroxide to water while oxidizing organic substrates and are valuable in spheres like industrial and medical applications and histochemistry. Limitations still exist in the use of the well-studied horseradish peroxidase for certain activities due to limitations like poor thermal stability, thus the search for novel peroxidases that can overcome these limitations is an active area of research. Butternut squash peroxidase (Cucurbita moschata) (BSP) shows promise due to significant activity being found in the skin and apparent enhanced thermal stability, but an efficient purification scheme for it is lacking, as well as …

Modeling And Synthesis Of 3-(2,6-Disubstituted 5-Pyrimidyl) Propionic Acids As Inhibitors Of Low Molecular Weight Protein Tyrosine Phosphatase (Lmw-Ptp), Kennedy Bueno, Madisen Carter

Celebrating Scholarship and Creativity Day (2018-)

Low molecular weight protein tyrosine phosphatase (LMW-PTP) is an enzyme and a known signal pathway for growth factors and cellular transformation in eukaryotic cells. Our research aims to synthesize 12 new potential inhibitors of LMW-PTP and analyze their inhibitory activity and binding affinity. The inhibition of both isoforms of LMW-PTP has been the primary focus of research due to their potential role in breast, colon, and other cancers as well as type II diabetes. One known inhibitor, pyridoxal 5’- phosphate (PLP), is essential for various enzymatic reactions within the body, such as the synthesis of neurotransmitters, and is therefore impractical …

Lc-Ms/Ms For Proteomic Analysis Of Post-Translational Modifications On Xct, Connor Bricco

23rd Annual A. Paul and Carol C. Schaap Celebration of Undergraduate Research and Creative Activity (2024)

Membrane protein xCT and its heavy chain component 4F2HC make up the xc- transport system. 4F2HC may be necessary for membrane localization of the heterodimer and xCT is responsible for transport activity. Under basal conditions, xCT resides in endosomes, but upon oxidative insult, xCT moves to the membrane and functions to reduce oxidative stress. We hypothesize the movement of xCT to the membrane is directed by changes in posttranslational modifications (PTMs) such as phosphorylation, ubiquitination and glycosylation. The overall goal of this project is to use mass spectrometry to detect the PTMs of xCT isolated from cells grown under basal …

Lysine 473 Regulates The Activity And Trafficking Of The Cystine/Glutamate Transporter, System XC-, Anna Koppin

23rd Annual A. Paul and Carol C. Schaap Celebration of Undergraduate Research and Creative Activity (2024)

System x_c^- is a membrane transport system that plays a critical role in mitigating oxidative stress. Past work in our lab has shown that System x_c^- localizes to the plasma membrane allowing for increased activity to support production of antioxidants during oxidative stress. In this study, we sought to determine if post-translational modification (PTM) of the transporter regulates its trafficking. A C-terminal 3KR mutant (K422,472,473R) exhibited decreased membrane localization and activity, suggesting that PTM at one of these sites increases activity. Further, we observed that K473R exhibits a 7 kD decrease in the molecular weight, indicating …

Ubiquitination Of Xct: Impacts On The Protein's Stability, Turnover Rate, And Localization, Sofia Rosenberger

23rd Annual A. Paul and Carol C. Schaap Celebration of Undergraduate Research and Creative Activity (2024)

System x_c^- imports cystine and exports glutamate. Its presence on the plasma membrane has been shown to increase directly with oxidative insults. Ubiquitin, a small protein, is directly involved in the trafficking and degradation of numerous proteins within cells and has been shown to bind to System x_c^-. Moreover, upon oxidative insult, protein ubiquitination increases. However, it is not understood how ubiquitination of the transporter impacts its activity. Therefore, the objective of this project is to directly assess how ubiquitination affects the protein’s stability, turnover rate, and localization in the context of oxidative stress. We …

Discovery Of A Small-Molecule Inhibitor That Traps Polθ On Dna And Synergizes With Parp Inhibitors, William Fried, Mrityunjay Tyagi, Leonid Minakhin, Gurushankar Chandramouly, Taylor Tredinnick, Mercy Ramanjulu, William Auerbacher, Marissa L Calbert, Timur Rusanov, Trung Hoang, Nikita Borisonnik, Robert Betsch, John Krais, Yifan Wang, Umeshkumar Vekariya, John Gordon, George Morton, Tatiana Kent, Tomasz Skorski, Neil Johnson, Wayne Childers, Xiaojiang Chen, Richard Pomerantz

Department of Biochemistry and Molecular Biology Faculty Papers

The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4-6 nM IC₅₀ that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in …

Parp2 Promotes Break Induced Replication-Mediated Telomere Fragility In Response To Replication Stress, Daniela Muoio, Natalie Laspata, Rachel L Dannenberg, Caroline Curry, Simone Darkoa-Larbi, Mark Hedglin, Shikhar Uttam, Elise Fouquerel

Department of Biochemistry and Molecular Biology Faculty Papers

PARP2 is a DNA-dependent ADP-ribosyl transferase (ARTs) enzyme with Poly(ADP-ribosyl)ation activity that is triggered by DNA breaks. It plays a role in the Base Excision Repair pathway, where it has overlapping functions with PARP1. However, additional roles for PARP2 have emerged in the response of cells to replication stress. In this study, we demonstrate that PARP2 promotes replication stress-induced telomere fragility and prevents telomere loss following chronic induction of oxidative DNA lesions and BLM helicase depletion. Telomere fragility results from the activity of the break-induced replication pathway (BIR). During this process, PARP2 promotes DNA end resection, strand invasion and BIR-dependent …