Biochemistry Commons^™

Structural Impacts Of Canonical And Non-Canonical Oligonucleotide Modifications, Deepak Shrestha

Wayne State University Dissertations

Nucleic acids, including DNA (deoxyribonucleic acid) and RNA (ribonucleic acids), are the fundamental building blocks of living organisms. The events that cause modification of DNA/RNA are of interest because they impact the nucleic acid function. Targeting DNA is important for treating diseases such as cancer. Cisplatin (cisPt) is a well-studied drug that targets DNA and blocks replication, but its use is limited by adverse side effects and cellular resistance. Prevoius research addressed some of these challenges through development of platinum analogues such as carboplatin and oxaliplatin. Amino acid-linked platinum analogues (AAPt) are the focus of this thesis work. AAPt complexes …

Mass Spectrometry Combined With Chemical Biology To Study Human Ailments, Eric Thomas Jacquerio Davis

Wayne State University Dissertations

Post-translational modifications (PTMs) are essential as chemical changes that regulate protein function, activity, stability, interactions, and localization after protein synthesis. PTMs, such as phosphorylation, acetylation, and ubiquitination, can activate or deactivate proteins, influence their interactions with other molecules, and determine their cellular location. This regulation is vital for numerous biological processes, such as cell cycle control, signal transduction, and gene expression. Abnormal PTMs are linked to various diseases in humans, including parasitic infection, blood disorders, cancers, and neurodegenerative diseases, making their study crucial for understanding disease mechanisms and developing targeted therapies. In the Pflum lab, we are interested in the …

Uracil Landscape Of Mouse B Cell Genome Undergoing Class-Switch Recombination, Fathima Rukshana Mohamad Ramshan

Wayne State University Dissertations

Activation-induced cytosine deaminase (AID) is required for both somatic hypermutation (SHM) and class switch recombination (CSR) within mammalian antibody genes. Uracils created by AID in the Ig loci are recognized and processed by base-excision and mismatch repair pathways leading to antibody diversification. This involves a hypermutation process restricted to the V(D)J segments of the immunoglobulin genes, SHM, and an error-prone end-joining process that causes isotype switching, CSR, but the molecular details of these processes have remained obscure for the past 20+ years. Most studies of SHM and CSR have used AID-promoted mutations as a proxy for the uracils, but their …

Design, Synthesis, And Evaluation Of Class Iia Histone Deacetylase (Hdac) Inhibitors, Lauren Nicole Kotsull

Wayne State University Dissertations

Histone deacetylase (HDAC) proteins are linked to disease states, including cancer, and have become important targets for therapeutic development. Many inhibitors have been developed to target HDAC proteins, including FDA-approved inhibitors Vorinostat (SAHA), Belinostat, Romidepsin, and Panobinostat. These inhibitors non-specifically influence most of the 11 metal-dependent HDAC isoforms, which might contribute to the side effects that patients experience during treatment. The lack of selectivity also diminishes the use of HDAC inhibitors as biomedical research tools. To address these drawbacks, the development of selective inhibitors has been heavily studied. Yet, reported inhibitors are predominantly selective for class I HDAC isoforms (HDACs …

Dual Mechanisms Contributing To Pyruvate Dehydrogenase Activity Deficiency In A Barth Syndrome Cell Model, Zhuqing Liang

Wayne State University Dissertations

Barth syndrome (BTHS) is a rare genetic disease that results from mutations in the TAFAZZIN gene, which encodes the cardiolipin (CL) remodeling enzyme tafazzin (Taz). The mechanisms linking perturbation of CL remodeling and the pathological features of BTHS are not understood. We have recently reported that intermediary metabolism is perturbed in BTHS, and activity of the metabolic gatekeeper enzyme pyruvate dehydrogenase (PDH) is deficient in BTHS models. The mechanism whereby PDH is regulated by Taz is unknown, and this knowledge gap represents an obstacle to the development of therapeutics to treat BTHS. Using an established C2C12 myoblast model of BTHS, …

Kinase-Catalyzed Labeling To Identify Kinase-Substrate Pairs Using Γ-Phosphate Modified Atp Analogs, Rachel Beltman

Wayne State University Dissertations

Post-translational modifications (PTMs) are responsible for a variety of cellular processes. One such PTM is protein phosphorylation, which is catalyzed by kinases. Kinase enzymes play important roles in cellular signaling pathways, but dysregulation of kinase-mediated events results in the formation of diseases, which make kinases favorable drug targets. To uncover the role kinases play in the development of diseases, kinase-mediated cellular events need to be better understood. The current gap in the field is the lack of tools available to identify the kinase that is responsible for specific phosphorylation events within the cell. To improve the gap in the field, …

Extending Plasma Proteome Coverage To Enable Biomarker Discovery, Jordan Bruce Burton

Wayne State University Dissertations

Two features make plasma a valued source of health information: First, plasma contains secreted proteins from all tissues in the body and can, therefore, provide information about the health status of all tissues. Second, plasma is available in large quantity using a minimally invasive procedure that is well tolerated. The status of plasma as a valued source of health information is matched by the difficulty in extracting proteome information from the samples. Using state of the art instrumentation a standard analysis of plasma yields quantitative information on approximately 300 proteins. Extreme fractionation strategies coupled with increased mass spectrometry time extends …

Identification Of Glutathionylated Cysteines Regulating Cell Migration, Dhanushika Kukulage

Wayne State University Dissertations

Cell migration is vital in many physiological processes, such as wound repair and metastasis. Impaired cell migration in wound healing may result in chronic wounds, by which 6.7 million Americans are affected, and they suffer from bacterial infections, amputations, increased mortality, and depression. Reactive oxygen species (ROS) or hydrogen peroxide (H2O2) play an essential role as a second messenger in redox signaling. Evidence has been found that H2O2 takes part in regulating cell migration as well. A primary mechanism of ROS regulating redox signaling is oxidative protein modifications. Protein S-glutathionylation, one of the oxidative modifications, is the conjugation of glutathione …

Characterizing The Post-Translational Modifications Of The Pro-Oncogenic Type Ii Transmembrane Serine Protease Tmprss13, Carly Elizabeth Martin

Wayne State University Dissertations

TMPRSS13, a type II transmembrane serine protease discovered at the turn of the century, has recently been shown to be significantly overexpressed in both breast cancer (BCa) and colorectal cancer (CRC), and to mediate chemoresistance in cell lines from both cancer types. Furthermore, loss of TMPRSS13 in a genetic model of mouse mammary carcinoma significantly reduced tumor burden and growth rate, and increased overall tumor-free survival. Its location on the cell surface as well as its low expression in normal breast and colon make TMPRSS13 an attractive candidate as an oncogenic biomarker and therapeutic target in cancer. However, little is …

Substrate Profiling Of The Epigenetic Erasers Hdac1 And Lsd1, Herath Mudiyansela Gedara Kavinda Eranga Herath

Wayne State University Dissertations

Regulators of chromatin structure have emerged as a key driver of transcriptional responses inside the cell. Two such groups of regulators, histone writers and erasers; the proteins, that add or remove histone post translational modifications (PTMs), have become the central players in chromatin structure. Thus, the aberrant expression of writers and erasures is a hallmark in human diseases. For example, overexpression of the erasures histone deacetylase1 (HDAC1) and lysine specific demethylase1 (LSD1) had been reported in many cancers. Currently, HDAC inhibitors have been used successfully for cancer treatment and several inhibitors targeting LSD1 are in clinical trial. To date, apart …

Developing Inhibitors Of Uchl5 Deubiquitinase In The Proteasome, Harshani Sewvandi Gurusingha Arachchige

Wayne State University Dissertations

Ubiquitin proteasomal system (UPS) plays a vital role in maintaining protein homeostasis by eliminating misfolded and damaged proteins in a eukaryotic cell. Target proteins to be degraded are modified by poly-ubiquitination, which leads to degradation by 26S proteasome. Before entering the 20S catalytic subunit, the 19S regulatory particle is responsible for binding, unfolding, and deubiquitinating the target protein. Based on the nature of the polyubiquitin tag, removal of the ubiquitin is performed by the deubiquitinases (DUBs) associated with the proteasome.Ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5) is one of the three DUBs in the proteasome. UCHL5 regulates many cellular functions and …

An In Vivo Inducible Expression System To Determine Antibacterial Activity Of Peptides, Rabiul Islam

Wayne State University Dissertations

The threat of bacterial resistance throughout the world has created tremendous challenges to clinicians. Some bacterial species have acquired resistance to not only single antibiotics, but also to multiple compounds, which is called multidrug resistance. Since the early discovery of resistance to penicillin, scientists have been developing new drugs to combat antibiotic resistance. In some cases, it took less than a decade to develop resistance to a new antibiotic. The World Health Organization (WHO) reported several deadly multi-drug-resistant bacteria, some of which cause life-threating illnesses. Each year more than 35,000 people in the United States die from antibiotic-resistant bacterial infections. …

Mnrr1: Understanding The Role Of A Novel Mitochondrial-Nuclear Regulator, Stephanie L. Gladyck

Wayne State University Dissertations

Mitochondria are complex organelles that generate most of the energy required to sustain life and function in metabolic and signaling pathways required to maintain cellular homeostasis. MNRR1 (mitochondrial nuclear retrograde regulator 1 or CHCHD2) is a small, bi-organellar twin CX9C protein that is emerging as an important regulator of mitochondrial function, apoptosis, and cellular stress by participating in mitochondrial-nuclear crosstalk. Our lab has previously shown that in the mitochondria, MNRR1 regulates complex IV (Cytochrome c oxidase or COX) and is able to finetune the oxidase function through phosphorylation status. We have also shown that during stress, mitochondrial MNRR1 levels deplete, …

Development And Application Of Chemical Tools To Identify Kinase-Substrate Interactions, Aparni Kithulgoda Gamage

Wayne State University Dissertations

Post translational modifications regulate a variety of biological processes inside the cell.Protein phosphorylation is one such PTM modification catalyzed by protein kinases, which aid to transfer a signal from one place to another inside the cell. However, irregularities in kinase-mediated signaling are often implicated in many diseases, making kinases effective drug targets. To understand kinase-related disease formation and to discover drugs to treat these diseases, it is crucial to have a clear understanding on kinase-mediated cell signaling networks. A current gap in the kinase biology field is a lack of tools to identify which kinase phosphorylates which protein substrate inside …

Variations On A Theme: Intricacies Of Unanchored Poly-Ubiquitin Signaling And Toxicity, Jessica Renee Blount-Pacheco

Wayne State University Dissertations

Ubiquitin is an 8.5 kDa post-translational modifier involved in essentially all eukaryotic cellular processes. Through a process called ubiquitination, ubiquitinating enzymes chemically attach ubiquitin to substrate proteins to control their fates, resulting in anything from their recruitment into signaling pathways to their proteasomal degradation, with a plethora of possibilities in between. Ubiquitin molecules can also be attached to one another, resulting in poly-ubiquitin chains with various effects depending on the number of ubiquitin molecules and the specific amino acid residues used to link them together. While most poly-ubiquitin in the cell exists as conjugated species, there are also untethered poly-ubiquitin …

Use Of Alkoxyamines To Quantify And Map Uracils And To Kill B Cell Cancers, Madusha Lakshani Watuthanthrige Perera

Wayne State University Dissertations

Majority of B-cell lymphoma and leukemia originates in germinal centers and over express AID resulting high levels of uracils and eventually AP sites. Treating cells with a small alkoxyamine AA3 with a terminal alkyne group specifically killed B cell lymphoma cell lines without killing normal human B cells and non-hematological cancers. To study the cytotoxicity of AA3, I designed and synthesized a series of AA3 analogs with and without the alkyne group. Using cytotoxicity assays I demonstrated that the alkyne functionality plays a major role in AA3 cytotoxicity in B-cell cancers and further showed that the effectiveness is modulated by …

Computational Analysis Of Oxidative Stress In Endothelial Dysfunction: Insights On The Role Of Tetrahydrobiopterin, Ascorbate And Glutathione, Sheetal Kedar Panday

Wayne State University Dissertations

Oxidative stress and endothelial dysfunction are reported in the cardiovascular and neurovascular diseases. Oxidative stress is caused due to an increase in the generation of reactive oxygen (ROS) and nitrogen species (RNS) and incapacity of antioxidant systems to eliminate ROS and RNS. Endothelial dysfunction is characterized by a reduction in nitric oxide (NO) bioavailability. NO is constitutively produced by enzyme endothelial nitric oxide synthase (eNOS). A reduction in tetrahydrobiopterin (BH4), which is an essential cofactor of eNOS, can lead to eNOS uncoupling. There is complex interplay between the ROS/RNS and antioxidant system underlying pathophysiologies of vascular diseases, however our quantitative …

Timing And Duration Of Folate Restriction Differentially Impacts Colon Carcinogenesis., Ali M. Fardous

Wayne State University Dissertations

Colorectal cancer (CRC) constitutes a major burden on the healthcare system as the second most commonly diagnosed cancer in the developed world. Dietary folate is considered an important modulator of colorectal cancer. Folate restriction has been implicated in increasing CRC incidence by disrupting nucleotide synthesis, Impacting DNA methylation and inducing genetic instability. Our research shows that the timing and duration of dietary folate restriction can differentially impact Colorectal cancer initiation. Acclimating mice to folate restriction for 8 weeks results in a reduced number of preneoplastic lesions compared to mice placed of folate restriction for 1 week prior to initiating the …

Termination-Independent Role Of Rat1 In Cotranscriptional Splicing In Budding Yeast, Zuzer Hakimuddin Dhoondia

Wayne State University Dissertations

Rat1 is a 5′→3′ exoribonuclease in budding yeast belonging to the XRN-family of nucleases. It is a highly conserved protein with homologs being present in fission yeast, flies, worms, mice and humans. Rat1 and its homolog in metazoan have been shown to function in multiple facets of RNA metabolism. In this study, we report a novel role of Rat1 in splicing of pre-mRNA in budding yeast. In the absence of the functional Rat1 in the nucleus, an increase in the level of unspliced transcripts was observed in yeast cells. Strand-specific TRO analysis revealed that the accumulation of unspliced transcripts upon …

Role Of Dyslipidemia On Lipid Metabolism In Maintenance Hemodialysis Patients, Eno Latifi

Wayne State University Dissertations

Maintenance hemodialysis (MHD) patients experience various abnormalities such as systemic inflammation (SI), oxidative stress (OS), and dyslipidemia (D). Defined as an imbalance of plasma lipids, lipoproteins, and lipid metabolism enzymes, D has been associated with a rise in morbidity and mortality within ESRD patients due to cardiovascular disease (CVD). However, the contribution of each of these parameters to D is poorly understood; moreover, the impact of the following parameters on dyslipidemia in different ethnicities is unknown. Hence, the objective of this study was to characterize D in a multi-ethnic cohort of ESRD patients. We hypothesized that the degree of dyslipidemia …

Trna Aminoacylation: New Protein Players And New Reactions, Whitney Noel Wood

Wayne State University Dissertations

TRNA AMINOACYLATION: NEW PROTEIN PLAYERS AND NEW REACTIONS

by

WHITNEY N. WOOD

May 2019

Advisor: Dr. Tamara L. Hendrickson

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

Protein translation must usually occur with high accuracy for an organism to survive. However, Helicobacter pylori, Staphylococcus aureus, and many other microorganisms including important human pathogens, lack one or more aminoacyl-tRNA synthetase (aaRS), the enzymes that typically aminoacylate tRNAs for ribosomal translation. These organisms must use an indirect pathway to aminoacylate some tRNAs. Specifically, H. pylori lacks the genes that encode for asparaginyl- and glutaminyl-tRNA synthetases (AsnRS and GlnRS, respectively). Instead, H. pylori uses …

Predicting The Structure And Selectivity Of Coiled-Coil Proteins, Mojtaba Jokar

Wayne State University Dissertations

A coiled-coil protein structure consists of two (in coiled-coil dimers) or more interacting α-helical strands that together form a left-handed supercoil structure. Many coiled-coil proteins are involved in significant biological functions such as the regulation of gene expression, known as transcription factors. Also coiled-coil structures entail unique mechanical properties critical to the function and integrity of various motor proteins, cytoskeletal filaments and extra-cellular matrix proteins. Engineering these transcription factors is also expected to create more efficient and practical solutions to treat neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and prion …

Insights Into Nucleic Acid-Platinum(Ii) Compound Interactions And Structural Impacts, Supuni Duneeshya Kamal Thalalla Gamage

Wayne State University Dissertations

With the discovery of cisplatin in the 1960s, it has been widely studied as a precursor for anticancer drug development. Despite its effectiveness against certain cancers, clinical usage of cisplatin is restricted by a number of side effects and resistance. In the past decade, scientists have been exploring biologically important ligands such as sugar derivatives in the hope of overcoming such challenges. Attachment of a sugar moiety could facilitate lower accumulation of platinum drugs in the body as well as enhance cellular uptake. In this study, a carbohydrate-linked cisplatin analog, cis-dichlorido[(2-β-D-glucopyranosidyl)propane-1,3-diammine]platinum (5) has been studied. The aim was to evaluate …

Functional Characterization Of Accessory Proteins And Novel Activities In Direct And Indirect Trna Aminoacylation, Udumbara Menike Rathnayake

Wayne State University Dissertations

Indirect tRNA aminoacylation is essential for most bacteria and archaea, particularly when these species do not have genes encoding asparaginyl- and/or glutaminyl-tRNA synthetase (AsnRS and GlnRS). In the absence of AsnRS, the first step in Asn-tRNAAsn synthesis involves misacylation of tRNAAsn with aspartate to produce Asp-tRNAAsn; this reaction is catalyzed by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS). Subsequently, in bacteria, an amidotransferase called GatCAB converts Asp-tRNAAsn to Asn-tRNAAsn. An analogous, two-step processes exist to produce Gln-tRNAGln. In this case, a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) misacylates tRNAGln to produce Glu-tRNAGln, which is then converted to Gln-tRNAGln by GatCAB. The central hub of …

Biochemical, Structural, And Drug Design Studies Of Aspartate Transcarbamoylase From Pseudomonas Aeruginosa And Staphylococcus Aureus, Chandni Patel

Wayne State University Dissertations

Sepsis affects 1.7 million people in the United States every year and nearly 270,000 people die as a result. Sepsis is characterized by systemic inflammation from an infection leading to organ dysfunction and death. Multi-drug resistance in bacteria is increasing globally, and Pseudomonas aeruginosa and Staphylococcus aureus are notorious for their multi-drug resistance and pose a serious need for the development of new antibiotics. The levels of pyrimidines in blood are too low to sustain the growth of bacteria, so they must rely on pyrimidine biosynthesis. Previous studies have shown that a defect in several pyrimidine biosynthetic enzymes resulted in …

Perturbation Of Energy Metabolism At The Center Of The Mechanism Of Action Of Valproate, Michael Ghassan Salsaa

Wayne State University Dissertations

Bipolar disorder (BD) is a common and debilitating psychiatric disorder. Valproic acid (VPA) is one of the major drugs used to treat BD patients. However, it is not universally effective and, in addition, causes severe side effects. Its mechanism of action is not known, which complicates efforts to develop more effective drugs. Studies have established that VPA perturbs metabolism, which is implicated in both the therapeutic mechanism of action of the drug as well as drug toxicity. However, the mechanism whereby VPA causes these perturbations is not understood. To address this knowledge gap, I investigated the acute and chronic effects …

The Dynamic Nature And Biophysical Characterization Of Isu1, Fe-S Cluster Assembly Scaffold Protein In Saccharomyces Cerevisiae, And Its Significance To Human Disease, Brianne Elizabeth Lewis

Wayne State University Dissertations

Mitochondrial Fe-S cluster biosynthesis is accomplished within yeast utilizing the biophysical characteristics of the “Isu1” scaffold protein. As a member of a highly homologous protein family, Isu1 has sequence conservation with orthologs and a conserved ability to assemble [2Fe-2S] clusters. Regardless of species, scaffold orthologs can exist in both “disordered” and “structured” conformations and is directly related to conformations utilized during Fe-cofactor assembly. During assembly, the scaffold directs the delivery and the utilization of both Fe(II) and sulfide substrates in order to produce [2Fe-2S] clusters, however Zn(II) binding can alter the activity of the scaffold with stabilizing the protein in …

Investigation Of The Saccharomyces Cerevisiae Gpi Transamidase: Insights Into Its Activity And Subunit-Subunit Interactions, Travis Ness

Wayne State University Dissertations

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a eukaryotic, posttranslational

modification catalyzed by GPI transamidase (GPI-T). The Saccharomyces

cerevisiae GPI-T is composed of five membrane-bound subunits: Gaa1, Gpi8, Gpi16,

Gpi17, and Gab1. Structural and functional studies have been hindered by the

complexity of this enzyme. Conditions to purify the Gpi8:Gaa1:Gpi16 GPI-T heterotrimer

from yeast have been reported, but an understanding of the subunit functions,

interactions, and stoichiometry remain unclear. Furthermore, a reliable, quantitative, in

vitro assay for this important post-translational modification has remained elusive for

nearly three decades.

Our laboratory has developed an in vitro peptide cleavage assay that correlates

changes …

Biochemical And Cellular Studies Of Apobec3 Family Dna-Cytosine Deaminases, Sachini Umedi Siriwardena

Wayne State University Dissertations

The AID/APOBEC family of enzymes deaminate cytosines in single-stranded DNA to uracils leading to base substitutions and strand breaks. Members of APOBEC3 family in humans are induced by cytokines produced during the body's inflammatory response to infections and provide innate immunity against viruses. However, there is emerging consensus that these enzymes can cause mutations in the cellular genome depending on the physiological state of the cell and the phase of the cell cycle they are expressed. Since aberrant expression of APOBEC3B was recently identified as a possible source of cancer, we initiated a study to determine the maximally active catalytic …

Design, Synthesis, And Reactivity Of Homo- And Heterobimetallic Complexes Bridged By A Xanthene Linker, Thilini Samangi Hollingsworth

Wayne State University Dissertations

Cooperative reactivity of bimettalics can be is observed in many different areas of chemistry and have been increasingly investigated because of the advantageous reactivity when compared to the corresponding mononuclear systems. The focus of my dissertation is on (1) investigation of the homobimetallic cooperativity in lactide polymerization catalysis; (2) investigation of the heterobimetallic cooperativity in the biomimetic studies of Mo-Cu carbon monoxide dehydrogenase (CODH) enzyme in order to make a functional model of its active site.

Three new main group bis(alkoxide) complexes Mg(OR)2(THF)2, Zn(Cl)(μ2-OR)2Li(THF) and In(OR)2(μ2-Cl)2Li(THF)2 featuring bulky alkoxide [OCtBu2Ph] were synthesized serve as metal alkoxide precursors for bimetallic lactide …