Biochemistry, Biophysics, and Structural Biology Commons^™

Molecular Insights Of Paraoxonase 2 In Advanced Glycation End Product Induced Endothelial Dysfunction, Ramya R

Theses and Dissertations

Diabetes mellitus (DM) is a metabolic disorder associated with various vascular complications and affects the function of the endothelial cells. Elevated blood glucose levels accelerate the formation of advanced glycation end products (AGEs) by Amadori rearrangement and bind to their Receptor for the advanced glycation end product (RAGE) and evoke release of key pro-inflammatory cytokine proteins leading to endothelial dysfunction. Paraoxonase 2 (PON2) is an endogenous intracellular enzyme, localised in mitochondria, endoplasmic reticulum (ER), and nuclear membrane with numerous properties, like anti-inflammatory, anti-apoptotic and anti-oxidant.

Our present study aims to ascertain the effect of AGEs on PON2 activity and expression, …

Using Edna To Assess Impacts Of Oyster Restoration On Ecosystem Biodiversity At A Heavily Impacted Coastal Lagoon, Dominique Di Domenico, Emily Bonacchi, Elizabeth Suter

Molloy Multidisciplinary Undergraduate Research Conference

The Great South Bay (GSB), a bar-built, temperate, coastal lagoon on the south shore of Long Island, New York, USA, is a highly modified estuary due to heavy urbanization and suburbanization in the last century. GSB historically provided up to 50% of the nation’s hard clam (Mercenaria mercenaria) harvest, and is the legal home of the “Blue Point” oyster (eastern oyster; Crassostrea virginica). However, due to overexploitation as well as persistent stress from ongoing nitrogen pollution and harmful algal blooms, shellfish populations are fractions of their original abundances. Restoration efforts in GSB, driven both by community efforts …

Trna Anticodon Cleavage By Target-Activated Crispr-Cas13a Effector, Ishita Jain, Matvey Kolesnik, Konstantin Kuznedelov, Leonid Minakhin, Natalia Morozova, Anna Shiriaeva, Alexandr Kirillov, Sofia Medvedeva, Alexei Livenskyi, Laura Kazieva, Kira S Makarova, Eugene V Koonin, Sergei Borukhov, Konstantin Severinov, Ekaterina Semenova

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA–guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus …

Predictive And Prognostic Biomarkers And Tumor Antigens For Targeted Therapy In Urothelial Carcinoma, Aditya Eturi, Amman Bhasin, Kevin Zarrabi, William Tester

Department of Medical Oncology Faculty Papers

Urothelial carcinoma (UC) is the fourth most prevalent cancer amongst males worldwide. While patients with non-muscle-invasive disease have a favorable prognosis, 25% of UC patients present with locally advanced disease which is associated with a 10-15% 5-year survival rate and poor overall prognosis. Muscle-invasive bladder cancer (MIBC) is associated with about 50% 5 year survival when treated by radical cystectomy or trimodality therapy; stage IV disease is associated with 10-15% 5 year survival. Current therapeutic modalities for MIBC include neoadjuvant chemotherapy, surgery and/or chemoradiation, although patients with relapsed or refractory disease have a poor prognosis. However, the rapid success of …

Upregulation Of The Predominant Cystic Fibrosis Causing Mutation Df508-Cftr By Triazole Compounds In Epithelial Cells, Maggie Taylor

Undergraduate Research Conference

Cystic fibrosis is a common genetic disease that is caused by a mutation in the plasma membrane protein CFTR, which stands for Cystic Fibrosis Transmembrane-conductance Regulator. When this membrane protein is mutated, it impairs its chloride ion channel function, blocking the movement of chloride ions that travel in and out of the cell. Previous studies have demonstrated that the most prevalent CFTR mutation, ∆F508-CFTR, can be partially reversed using small molecules (Heda and Marino, BBRC, 271:659-664, 2000). In this study, I have investigated the effects of several triazole compounds known to bind and transport chloride ions in cultured cells, on …

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 …

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 …

Multivalency Of The Mdmx Acidic Domain Slims Use Different Binding Modes To Inhibit Mdmx And P53, Malissa Fenton

USF Tampa Graduate Theses and Dissertations

Regulation of protein binding through autoinhibition commonly occurs via interactions involving intrinsically disordered regions (IDRs). These intramolecular interactions can directly or allosterically inhibit intermolecular protein or DNA binding, regulate enzymatic activity, and control the assembly of large macromolecular complexes. Autoinhibitory interactions mediated by protein disorder are inherently transient, making their identification and characterization challenging. In this work, we explore the structural and functional diversity of disorder-mediated autoinhibition for a variety of biological mechanisms, with a focus on the role of multivalency and effective concentration. We also discuss the evolution of disordered motifs that participate in autoinhibition using examples where sequence …

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 …

Social Media Does Not Elicit A Physiological Stress Response As Measured By Heart Rate And Salivary Cortisol Over 20-Minute Sessions Of Cell Phone Use, Suzanne Oppenheimer, Laura Bond, Charity Smith

Biomedical Research Institute Publications and Presentations

The pervasive use of social media has raised concerns about its potential detrimental effects on physical and mental health. Others have demonstrated a relationship between social media use and anxiety, depression, and psychosocial stress. In light of these studies, we examined physiological indicators of stress (heart rate to measure autonomic nervous system activation and cortisol to assess activity of the hypothalamic-pituitary-adrenal axis) associated with social media use and investigated possible moderating influences of sex, age, and psychological parameters. We collected physiological data from 59 subjects ranging in age from 13 to 55 across two cell phone treatments: social media use …

The Impact Of Mutations In The Arabidopsis Apetela (Ap3) Gene, Hazel R. Frans, Tara Phelps-Durr

SACAD: Scholarly Activities

The purpose of this research is to understand the molecular functioning of the Arabidopsis thaliana Apetela (Ap3) gene. We created mutations in two sites of the gene, AP3-3 and AP3-5. These are predicted to change AP3 protein structure, which may result in a mutated flower. Analyzing the effects of new mutations allows an understanding of protein formation both in plants and humans.

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 …

Maldi-Msi Identification Of Tissue-Level N-Glycomic And Proteomic Molecular Biomarkers Of Aggressive Prostate Cancer, Jordan Paige Hartig

MUSC Theses and Dissertations

Prostate Cancer (PCa) poses a significant clinical challenge, characterized by debates on screening efficacy and continuous issues with distinguishing aggressive from non-aggressive tumors. Despite treatment advancements, poor patient outcome at late stages and clinical overtreatment of indolent disease emphasizes the urgent need for markers of PCa aggression. Aberrant glycosylation and extracellular matrix (ECM) remodeling are two complex molecular processes necessary for PCa progression, highlighting a novel area for biomarker discovery. Leveraging formalin-fixed paraffin-embedded tissues, tissue microarrays (TMAs), and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), distinct N-glycan and ECM profiles are identified. Elevated bisecting, multi-antennary, and fucosylated N-glycans indicate early …

In Silico Identification Of Small Molecule Agonist Binding Sites On Kcc2, Kenyon Mitchell, Alfred Amendolara, Ruth Hunter, Jaden Miner, Andrew Payne

Annual Research Symposium

Purpose: Potassium-Chloride Cotransporter 2 (KCC2) is a neuronal membrane protein specific to the central nervous system. It is responsible for removing Cl- ions from the intracellular space, maintaining a normal Cl- gradient essential for proper function at inhibitory synapses. Dysregulation causes an upward shift in the Cl- reversal potential resulting in a hyperexcitable state of the postsynaptic neuron. Existing literature indicates that KCC2 may be involved in the addiction pathway of a variety of drugs of abuse, including opioids and alcohol. This makes KCC2 an attractive potential drug target when treating substance use disorders. A novel direct KCC2 agonist, VU0500469, …

Neurotensin Modulates Ovarian Vascular Permeability Via Adherens Junctions, Andrew Pearson, Ketan Shrestha, Thomas E. Curry, Diane M. Duffy

UK CARES Faculty Publications

Neurotensin (NTS) is a 13-amino acid peptide which is highly expressed in the mammalian ovary in response to the luteinizing hormone surge. Antibody neutralization of NTS in the ovulatory follicle of the cynomolgus macaque impairs ovulation and induces follicular vascular dysregulation, with excessive pooling of red blood cells in the follicle antrum. We hypothesize that NTS is an essential intrafollicular regulator of vascular permeability. In the present study, follicle injection of the NTS receptor antagonist SR142948 also resulted in vascular dysregulation. To measure vascular permeability changes in vitro, primary macaque ovarian microvascular endothelial cells (mOMECs) were enriched from follicle aspirates …

Tools For Biomolecular Modeling And Simulation, Xin Yang

Mathematics Theses and Dissertations

Electrostatic interactions play a pivotal role in understanding biomolecular systems, influencing their structural stability and functional dynamics. The Poisson-Boltzmann (PB) equation, a prevalent implicit solvent model that treats the solvent as a continuum while describes the mobile ions using the Boltzmann distribution, has become a standard tool for detailed investigations into biomolecular electrostatics. There are two primary methodologies: grid-based finite difference or finite element methods and body-fitted boundary element methods. This dissertation focuses on developing fast and accurate PB solvers, leveraging both methodologies, to meet diverse scientific needs and overcome various obstacles in the field.

A Comparison Of In Vitro Studies Between Cobalt(Iii) And Copper(Ii) Complexes With Thiosemicarbazone Ligands To Treat Triple Negative Breast Cancer, Duaa R. Alajroush, Chloe B. Smith, Brittney F. Anderson, Ifeoluwa T. Oyeyemi, Stephen J. Beebe, Alvin A. Holder

Undergraduate Research Symposium

Triple negative breast cancer (TNBC) is one of the most aggressive forms of breast cancer, and disproportionately affects African American women. TNBC cells lack the common hormone receptors that many pre-existing cancer treatments target. Fortunately, metal-based complexes with thiosemicarbazone ligands have gained significant attention for their potential as anti-cancer agents. Cobalt(III) complex ([Co(phen)2(MeATSC)](NO3)3•1.5H2O•C2H5OH]) and Copper(II) complex ([Cu(acetylethTSC)Cl]Cl•0.25C2H5OH) specifically have properties of high toxicity, which can contribute to decreased cancer cell activity. The effects of these complexes are currently being investigated on cancerous and non-cancerous breast cell lines. The cytotoxic effect of the cobalt(lll) complex and the copper(ll) complex was analyzed …

Myeloid-Derived Suppressor Cell Mitochondrial Fitness Governs Chemotherapeutic Efficacy In Hematologic Malignancies, Saeed Daneshmandi, Jee Eun Choi, Qi Yan, Cameron R. Macdonald, Manu Pandey, Mounika Goruganthu, Nathan Roberts, Prashant K. Singh, Richard M. Higashi, Andrew N. Lane, Teresa W-M Fan, Jianmin Wang, Philip L. Mccarthy, Elizabeth A. Repasky, Hemn Mohammadpour

Markey Cancer Center Faculty Publications

Myeloid derived suppressor cells (MDSCs) are key regulators of immune responses and correlate with poor outcomes in hematologic malignancies. Here, we identify that MDSC mitochondrial fitness controls the efficacy of doxorubicin chemotherapy in a preclinical lymphoma model. Mechanistically, we show that triggering STAT3 signaling via β2-adrenergic receptor (β2-AR) activation leads to improved MDSC function through metabolic reprogram- ing, marked by sustained mitochondrial respiration and higher ATP generation which reduces AMPK signaling, altering energy metabolism. Furthermore, induced STAT3 signaling in MDSCs enhances glutamine consumption via the TCA cycle. Metabolized glutamine generates itaconate which downregulates mitochondrial reactive oxygen species via regulation of …

Blocking The Dimerization Of Polyglutamine-Expanded Androgen Receptor Protects Cells From Dht-Induced Toxicity By Increasing Ar Turnover, Allison Lisberg, Yuhong Liu, Diane E. Merry

Department of Biochemistry and Molecular Biology Faculty Papers

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular degenerative disease caused by a polyglutamine expansion in the androgen receptor (AR). This mutation causes AR to misfold and aggregate, contributing to toxicity in and degeneration of motor neurons and skeletal muscle. There is currently no effective treatment or cure for this disease. The role of an interdomain interaction between the amino- and carboxyl-termini of AR, termed the N/C interaction, has been previously identified as a component of androgen receptor-induced toxicity in cell and mouse models of SBMA. However, the mechanism by which this interaction contributes to disease pathology is unclear. …

Biomarkers For Managing Neurodegenerative Diseases, Lara Cheslow, Adam E. Snook, Scott A. Waldman

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

Neurological disorders are the leading cause of cognitive and physical disability worldwide, affecting 15% of the global population. Due to the demographics of aging, the prevalence of neurological disorders, including neurodegenerative diseases, will double over the next two decades. Unfortunately, while available therapies provide symptomatic relief for cognitive and motor impairment, there is an urgent unmet need to develop disease-modifying therapies that slow the rate of pathological progression. In that context, biomarkers could identify at-risk and prodromal patients, monitor disease progression, track responses to therapy, and parse the causality of molecular events to identify novel targets for further clinical investigation. …

In Silico Analysis Of C-Type Lectins As Co-Infection Receptors Of Dengue And Chikungunya Viruses In Aedes Aegypti, Munawir Sazali, R. C. Hidayat Soesilohadi, Nastiti Wijayanti, Tri Wibawa, Arif Nur Muhammad Ansori

Makara Journal of Science

Aedes aegypti is a primer vector of dengue virus (DENV) and chikungunya virus (CHIKV). The susceptibility of mosquitoes to DENV and CHIKV depends on their recognition receptor of pathogens. C-type lectins (CTLs) are an important mediator of virus infection in A. aegypti. This study aims to identify potential receptors and determine the binding affinity between ligand–receptor interaction, CTLs and virus envelopes (DENV-1, 2, 3, and 4 and CHIKV) interaction based on in silico analysis. Sample sequences were obtained from GenBank (NCBI), and 10 CTLs were acquired from VectorBase. Homology modeling based on a minimum standard of 20% was processed …

Trip13’S Crucial Role In Pancreatic Cancer Progression, Swati Dhasmana, Anupam Dhasmana, Stella Rios, Iris A. Enriquez-Perez, Sheema Khan, Farrukh Afaq, Upender Manne, Murali M. Yallapu, Subhash Chauhan

Research Symposium

Background: Pancreatic cancer, characterized by its high mortality rate, stands as one of the most aggressive cancer forms. The projected surge in pancreatic cancer-related deaths, making it the second leading cause in the United States by 2030, underscores the urgency for effective early screening tools. This study employs data mining methods to scrutinize bioinformatic data surrounding TRIP13. Examining differential expression across various cancers, correlating TRIP13 expression with pancreatic cancer stages, exploring associations with common cancer genes, and analyzing overall survival rates constitute the core investigations. Integrated with molecular biology techniques, the study further quantifies TRIP13 expression in progressive pancreatic cancer …

Targeting Mycobacterial Efflux System For Combating Anti-Microbial Resistance, Arathi Radhakrishnan, Raj Kishor Kapardar, Rajpal Srivastav

Research Symposium

Background: The drug resistance in the microbes is a serious concern in medicine. Along with intrinsic factors, extrinsic factors like unprescribed usage of drugs are the contributing factors. The drug tolerance has led to the emergence of superbugs. Mycobacterial species utilize an array of multidrug efflux mechanisms linked to intrinsic and acquired antibiotic resistance. Understanding molecular mechanisms regulating efflux could reveal new therapeutic targets and strategies. Our study is aimed to target regulators of efflux Mycobacterial transporter.

Methods: Using the reference mycobacterial strain, antibiotic sensitivity was first profiled by minimal inhibitory concentration assays across a panel of antimicrobials, followed by …

Proteomic Analysis Of Stress Associated Factor Overexpression In Hepatocellular Carcinoma, Mohammad Shabir Hussain, Sophia Leslie, Amayrani Sanchez, Samantha Lopez, Kyle Doxtater, Manish Kumar Tripathi

Research Symposium

Background: Hepatocellular carcinoma (HCC) constitutes a substantial portion, accounting for 85% to 90% of liver cancers worldwide. Notably, within the Hispanic population, liver cancer mortality rates are notably higher, particularly evident in regions like the South Texas Rio Grande Valley (RGV), where nearly 90% of the populace is Latino/Hispanic. This region grapples with poverty affecting nearly 30% of its residents, coupled with elevated rates of obesity, diabetes, and low-income households, thereby fostering a prevalent environment of stress. Stress can profoundly impact cancer outcomes by compromising immune functionality and triggering inflammatory responses, potentially impairing surveillance against oncogenic triggers. The activation of …

Piperlongumine Nanoformulation Attenuates Pancreatic Tumor Desmoplasia And Alter Tumor Immune Responses, Vivek Kumar Kashyap, Neeraj Chauhan, Mohammed Sikander, Eswara N. H. K. Ghali, Bilal Hafeez, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan

Research Symposium

Pancreatic cancer (PanCa) is characterized by lack of early diagnosis, poor response to available therapeutic modalities and chemoresistance. Gemcitabine (GEM) is currently considered the most effective therapy for PanCa; however, it shows only a marginal survival benefit of 6 months. This poor drug response has been attributed to desmoplasia, causes suboptimal drug delivery, alters tumor microenvironment (TME), which includes tumor surrounding blood vessels, fibroblasts, immune cells, extracellular matrix, and other signaling molecules and induces chemo-resistance in tumors. To overcome these existing issues associated with chemotherapy, identification and development of novel therapeutic modalities are a pressing need. Piperlongumine (PL) is a …

Is Edna Concentration In Staphylococcus Aureus Biofilm Affected By Nanotube Coating?, Brooklyn Z. Jones, Bradford K. Berges, Lucy C. Bowden

Library/Life Sciences Undergraduate Poster Competition 2024

1st Place Oral Presentation

• ~30% of artificial joint failure is caused by infection1

• 80-100% of patients who receive external fixator pins will experience an infection2

• Most of these infections are caused by Staphylococcus aureus

• S. aureus forms biofilms (surface-associated collections of bacteria) that are extremely difficult to treat

• eDNA provides structure and protection to the biofilm

Using Chimeric Autoantigen Receptor (Caar) T Cells To Eliminate Autoreactive B Cells In Autoimmune Diseases, Abigail Cheever, Chloe Kang, Hunter Lindsey, Mackenzie Hansen, Kim O'Neill, Scott Weber

Library/Life Sciences Undergraduate Poster Competition 2024

• Graves’ disease (GD) is an antibody-mediated autoimmune disease affecting the thyroid and causes hyperthyroidism
• 4th most common autoimmune disease in the US
• Autoreactive B cells produce anti-TSHR antibodies (TRAbs) which mimic thyroid stimulating hormone (TSH) and chronically stimulate thyroid cells causing hyperthyroidism
• There currently is no treatment which addresses the root cause of the disease

Effects Of Post-Translational Histone Modifications On Transcription Rate, Jacob Hunter, Steven Johnson, Aaron Bohn, Sarah Ricks, Sarah Hodson, David Bates, Steven M. Johnson

Library/Life Sciences Undergraduate Poster Competition 2024

DNA structure and organization in eukaryotic cells significantly impacts the regulation of geneexpression, cellular properties and overall behavior. At the most fundamental unit of this organization,~147 bp of DNA wraps 1.7 times around a histone octamer core, forming a collective unit called thenucleosome. Positioning and occupancy of nucleosomes around promoter elements of genes is knownto be a strong regulator of transcription in eukaryotic nuclei. Post-translational modifications (PTM’s) tothe protruding N-terminal tails of histone proteins are known to influence chromatin structure and thusgene expression; however, relatively little is known about the residual effect of histone PTM’s ontranscription rate.

Two Dot1 Enzymes Cooperatively Mediate Efficient Ubiquitin-Independent Histone H3 Lysine 76 Tri-Methylation In Kinetoplastids, Victoria Frisbie, Hideharu Hashimoto, Yixuan Xie, Francisca De Luna Vitorino, Josue Baeza, Tam Nguyen, Zhangerjiao Yuan, Janna Kiselar, Benjamin Garcia, Erik Debler

Department of Biochemistry and Molecular Biology Faculty Papers

In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 through histone H2B mono-ubiquitin interaction, while the kinetoplastid Trypanosoma brucei di-methyltransferase DOT1A and tri-methyltransferase DOT1B efficiently methylate the homologous H3K76 without H2B mono-ubiquitination. Based on structural and biochemical analyses of DOT1A, we identify key residues in the methyltransferase motifs VI and X for efficient ubiquitin-independent H3K76 methylation in kinetoplastids. Substitution of a basic to an acidic residue within motif VI (Gx₆K) is essential to stabilize the DOT1A enzyme-substrate complex, while substitution of the motif X sequence VYGE by CAKS renders a rigid active-site …