Physical Sciences and Mathematics Commons^™

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, …

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 …

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 …

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 …

Functional Study Of Smyd2 Glutathionylation In Cardiomyocytes, Dhanushka Nalin Perera Munkanatta Godage

Wayne State University Dissertations

Reactive oxygen species (ROS) are important signaling molecules that contribute to the etiology of multiple muscle-related diseases, including cardiomyopathy and heart failure. There is emerging evidence that cellular stress can lead to destabilization of sarcomeres, the contractile unit of muscle. However, it is not completely understood how cellular stress or ROS induce structural destabilization of sarcomeres or myofibrils. Protein glutathionylation is one of the major protein cysteine oxidative modifications that play an important role in redox signaling and oxidative stress. In this report, we used a clickable glutathione approach in a cardiomyocyte cell line, and found that SET and MYND …

Carbohydrate-Based Inducers Of Cellular Stress For Targeting Cancer Cell Metabolism, Fidelis Ndombera

Wayne State University Dissertations

ABSTRACT

CARBOHYDRATE-BASED INDUCERS OF CELLULAR STRESS FOR TARGETING CANCER CELL METABOLISM

by

FIDELIS TOLOYI NDOMBERA

May 2018

Advisor: Dr. Young-Hoon Ahn

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

Metabolic reprogramming and redox control of cancer cells is vital for their proliferation, but also provides selective strategies for treating cancer. Increased generation of reactive oxygen species (ROS) and an intricate control of redox status in cancer cells relative to normal cells provide a basis for designing ROS-inducing anticancer agents. In my work, I designed, synthesized and evaluated carbohydrate-based small molecules for ROS-generation, cytotoxicity and redox signaling and stress response. Our data …

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 …

The Development Of Chemical Methods To Discover Kinase Substrates And Map Cell Signaling With Gamma-Modified Atp Analog-Dependent Kinase-Catalyzed Phosphorylation, Dissanayaka Mudiyanselage Maheeka Madhubashini Embogama

Wayne State University Dissertations

Kinase-catalyzed phosphorylation plays an important role in cell physiology by regulating a myriad of cellular functions. Thus aberrant kinase activity is implicated in various diseases. Methods are needed to discover kinase substrates and map signaling pathways to explore biology and to help drug discovery. A few techniques are currently available to discover kinase substrate and map cell signaling. However, to augment kinase substrate discovery approaches, it is essential to develop alternative techniques. Pflum has recently discovered cosubstrate promiscuity of protein kinases with gamma-modified ATP analogs. Here, kinase-catalyzed biotinylation with ATP-biotin was used to develop novel tools to discover kinase substrates …

Design, Synthesis And Biological Evaluation Of Histone Deacetylase (Hdac) Inhibitors: Saha (Vorinostat) Analogs And Biaryl Indolyl Benzamide Inhibitors Display Isoform Selectivity, Ahmed Negmeldin

Wayne State University Dissertations

HDAC proteins have emerged as interesting targets for anti-cancer drugs due to their involvement in cancers, as well as several other diseases. Several HDAC inhibitors have been approved by the FDA as anti-cancer drugs, including SAHA (suberoylanilide hydroxamic acid, Vorinostat). Unfortunately, SAHA inhibits most HDAC isoforms, which limit its use as a pharmacological tool and may lead to side effects in the clinic. In this work we were interested in developing isoform selective HDAC inhibitors, which may decrease or eliminate the side effects associated with non-selective inhibitors treatment. In addition, isoform selective HDAC inhibitors can be used as biological tools …

Development Of Chemical Tools To Investigate Protein S-Glutathionylation In Response To Metabolic Alteration, Kusal Theekshana Gayan Samarasinghe

Wayne State University Dissertations

Oxidative stress is a common characteristic of age-related diseases such as vascular diseases, diabetes and cancer. Many diseases are known to be regulated by glutathionylation. Glutathionylation is referred to as the formation of disulfide bond between a protein cysteine and a glutathione. To understand the molecular mechanisms behind the disease initiation and progression, identification of such glutathionylated proteins is important. Even though existing methods have been widely used, several limitations of these methods hinder the identification of such proteins in disease conditions. Therefore, we developed a versatile chemical method that generates clickable glutathione inside the cells. In this method, we …

Real-Time Investigation Of Bulky Lesion Bypass By Y-Family Dna Polymerase, Dpo4, Using Single Molecule Fret, Pramodha Liyanage

Wayne State University Dissertations

DNA is constantly exposed to various DNA damaging agents that are generated by various internal and external sources. Some of this damage may not be able to be repaired by cellular machineries causing DNA replication to be blocked. Once the replication fork is blocked by a DNA adduct, damage tolerance DNA polymerases, mainly Y-family, are able to restore the DNA replication by synthesizing past the DNA adduct. Benzo[a]pyrene (B[a]P) is one of the most studied environmental carcinogens. It is known to make covalent DNA adducts after metabolic activation and the bulkiness of the B[a]P adducts impose a strong barrier to …

Ligand Binding Studies Of A Peptide Targeting Helix 69 Of 23s Rrna In Bacterial Ribosomes, Hyosuk Seo

Wayne State University Dissertations

In the development of finding a peptide targeting H69 of 23S rRNA in bacterial ribosomes, phage display was employed at pH 5.5, a buffer condition previously reported of H69 preferring a closed conformation. After sequencing, several peptides were chosen through sequence alignment, followed by preparation using solid-phase peptide synthesis. The peptides were characterized using MALDI-TOF and purified with HPLC. A truncated peptide TARHIY was selected from FID assay. Through binding studies using ESI-MS, SPR, BLItz, and NMR, the binding properties of the peptide to H69 were determined, such as binding affinity, stoichiometry, and interaction site. The peptide exhibited moderate binding …

Development Of Gamma-Modified Atp Analogs To Study Kinase-Catalyzed Phosphorylations, Ahmed Eid Fouda

Wayne State University Dissertations

Kinase-catalyzed protein phosphorylation is one of the most important post-translational modifications that controls cascades of biochemical reactions. Irregularities in phosphorylation result in many diseases, such as diabetes mellitus, Parkinsons, and cancer. The development of new methods to monitor kinase-catalyzed phosphorylation is needed to decipher details of normal and diseased cell signaling. The Pflum lab recently developed several -modified ATP analogs to study kinase catalyzed phosphorylation reactions. The -modified ATP analogs have different tags, such as biotin for substrate labeling or aryl-azide for kinase substrates identification. Unfortunately, use of -modified ATP analogs was limited to in vitro studies due to the …

The Development Of Peptide Ligands To Target H69 Rrna, Danielle Nicole Dremann

Wayne State University Dissertations

ABSTRACT

THE DEVELOPMENT OF PEPTIDE LIGANDS TO TARGET H69

by

DANIELLE NICOLE DREMANN

December 2015

Advisor: Prof. Christine S. Chow

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

In the development of peptide ligands to target H69, SPPS and ESI MS was used to determine if 1) peptides could bind to modified H69 and 2) if increased affinity for the target RNA could be enhanced with modification. An alanine and arginine scan was synthesized and tested for this determination. Selected peptides were then tested using biophysical techniques such as circular dichroism and isothermal titration calorimetry. An assay was also designed to …

Dna Aptamers Selected Against Wild-Type Helix 69 Ribosomal Rna And Their Implications In Combating Antibiotic Resistance, Sakina Miriam Hill

Wayne State University Dissertations

Outbreaks of advanced antibiotic-resistant strains of microbes have hastened the need to identify new viable molecular targets for the development of novel anti-infectives. For this purpose, helix 69 (H69, or m3a 19-nucleotide (nt) hairpin motif that is highly conserved throughout phylogeny and rich in modified nucleotides, including pseudouridine () and 3-methylpseudouridine (m3) was chosen as a potential target. Helix 69, which is located in domain IV of Escherichia coli 23S ribosomal RNA (rRNA), undergoes conformational changes when in close proximity to the decoding region of 16S rRNA and transfer RNAs (tRNAs) in the peptidyl-transferase center (PTC). Functionally, the exact biological …

Investigation Into The Binding Interactions Of Klenow Fragment To Dna Modified With Carcinogens Af And Aaf Using Surface Plasmon Resonance, Ashley M. Floyd

Wayne State University Dissertations

The two major forms of DNA adducts from the carcinogen N-acetoxyacetyl-2-aminofluorene, N-(deoxygunanonsin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), are both known to impede replication, though in different ways. AAF is a strong block to replication leading to frameshift mutations, while the AF adduct is more easily bypassed, causing base substitutions. Surface plasmon resonance (SPR) was used to study the binding of exonuclease deficient E. coli polymerase I, Klenow fragment (KF), to DNA modified with AF or AAF at two locations: as a templating base or in the last formed base pair. KF binding to the modified DNA bases was also monitored to …

Characterization Of Initial Iron Binding Location And The Structure/Iron Binding Site On S.Cerevisiae Isu And On D.Melanogaster Frataxin, Andria V. Rodrigues

Wayne State University Dissertations

Iron-induced free radical damage has been implicated in the pathology of diseases of iron overload such as Friedreich's Ataxia, a genetic disorder characterized by an accumulation of iron in actively metabolizing tissues ultimately leading to cardio- and neuro- degeneration and cell death. It is caused by an inability to synthesize the mitochondrial protein, frataxin. Frataxin has been shown by numerous groups to be a part of the iron-sulfur cluster (ISC) multicomplex, where it functions in the capacity of a potential iron provider and an allosteric modulator of both the cysteine desulfurase and scaffold protein ISU. My research has been focused …

Structural And Functional Characterization Of Proteins Of Unknown Function (Hp0495, Hp0100 And Hp1259) In Helicobacter Pylori, Shirin Fatma

Wayne State University Dissertations

H. pylori is missing the glutaminyl- and asparaginyl-tRNA synthetases (GlnRS and AsnRS, respectively). Consequently, H. pylori uses an indirect aminoacylation pathway to generate Gln-tRNAGln and Asn-tRNAAsn. Within this process, Asn-tRNAAsn is produced by misacylation of tRNAAsn with aspartate by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS). Next, the heterotrimeric, glutamine-dependent amidotransferase (called AdT or GatCAB) converts the misacylated Asp-tRNAAsn into Asn-tRNAAsn. A parallel pathway exists for the synthesis of Gln-tRNAGln, wherein misacylation of tRNAGln with glutamate is catalyzed by a tRNAGln-specific glutamyl-tRNA synthetase (GluRS2) to generate Glu-tRNAGln; this misacylated intermediate is converted to Gln-tRNAGln by the same AdT. This dependence on misacylated …

Assembly And Function Of Macromolecular Complexes For Accurate Trna Aminoacylation In Helicobacter Pylori, Gayathri Niroshani Silva

Wayne State University Dissertations

ABSTRACT

ASSEMBLY AND FUNCTION OF MACROMOLECULAR COMPLEXES FOR ACCURATE TRNA AMINOACYLATION IN HELICOBACTER PYLORI

by

GAYATHRI SILVA

January 2014

Advisor: Dr. Tamara L. Hendrickson

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

Abstract

The aminoacylation of tRNA is a critical step in maintaining the accuracy of the genetic code. Many microorganisms are missing one or more aminoacyl tRNA synthetases (aaRSs) and rely on indirect pathways to produce certain aa–tRNAs. In Helicobacter pylori (H. pylori), the genes encoding both asparaginyl tRNA synthetase (AsnRS) and glutaminyl tRNA synthetase (GlnRS) are missing and the organism consequently relies on the indirect pathway for …

Investigating Hfq-Mrna Interactions In Bacteria, Martha Audra Faner

Wayne State University Dissertations

Regulatory RNAs (sRNAs) are essential for bacteria to thrive in diverse environments and they also play a key role in virulence [11]. Trans-sRNAs affect the stability and/or translation of their target mRNAs through complementary base-pairing. The base-pairing interaction is not perfect and requires the action of an RNA binding protein, Hfq. Hfq facilitates these RNA-RNA interactions by stabilizing duplex formation, aiding in structural rearrangements, increasing the rate of structural opening, and/or by increasing the rate of annealing [18-21]. Hfq has two well characterized binding surfaces: the proximal surface, which binds AU rich stretches typical of sRNAs, and the distal surface, …

Single-Molecule Studies Of Local And Global Nucleic-Acid Dynamics, Eric Muthuri Patrick

Wayne State University Dissertations

Nucleic acids undergo both global and local conformational changes that are important for their function. Structural studies have over the decades been invaluable in elucidation of various biomolecular mechanisms, hence contributing significantly to the understanding of biological events. However, a clear understanding of how molecules function in the cellular context requires investigation of their interconversion between multiple conformations, including mapping the folding landscape and any coupled changes in conformation. Work in this thesis focuses on fluorescence experiments, mainly at a single-molecule level to investigate such processes.

First, a novel single-molecule approach is described focusing on local dynamics within nucleic acids …

Development Of Peptide Inhibitors Targeting Clostridium Difficile Toxins A/B And Characterizing The Regulatory Role Of A Putative Negative Regulator Tcdc In Clostridium Difficile Toxin Gene Expression, Sanofar Jainul Abdeen

Wayne State University Dissertations

Clostridium difficile infections cause one of the most common and vital hospitalacquired

diseases often associated with broad-spectrum antibiotic usage. TcdA and TcdB

are the key virulence factors involved in major patho-physiology. While standard

antibiotics provide some respite, due to the high relapse rates and the emergence of more

severe disease presentations, antibiotics alone have often proven to be suboptimal.

Therefore there is a desperate need to develop an effective non-antimicrobial

therapeutics. Part of this work focuses on identification and further characterization of

peptide therapeutic that target the major virulence factor TcdA/TcdB. Towards

development of mechanistic-based anti-toxin agent, phage display was …

Development And Optimization Of The First High Throughput In Vitro Fret Assay To Characterize The Saccharomyces Cerevisiae Gpi-T, Sandamali Amarasingha Ekanayaka

Wayne State University Dissertations

DEVELOPMENT AND OPTIMIZATION OF AN IN VITRO FRET ASSAY TO CHARACTERIZE THE SACCHAROMYCES CEREVISIAE GPI TRANSAMIDASE

By

SANDAMALI AMARASINGHA EKANAYAKA

December 2013

Advisor: Dr. Tamara L. Hendrickson

Major: Biochemistry

Degree: Doctor of Philosophy

The enzyme glycosylphosphatidylinositol transamidase (GPI-T) mediates the attachment of a glycosylphosphatidylinositol (GPI) anchor to the C-terminus of specific proteins to produce GPI anchored proteins. This post-translational modification is essential for viability of eukaryotic organisms. However, very little is known about GPI-T and its catalytic activity. Thus, the research described in this abstract was conducted to develop an in vitro assay to monitor GPI-T. A high-throughput assay for …

Synthesis And Application Of Atp Analogs For Phosphorylation-Dependent Kinase-Substrate Crosslinking, Satish Kumar Garre Venkata Raghavendra

Wayne State University Dissertations

Phosphorylation is an important post-translational modification that plays a key role in a variety of signaling cascades and cellular functions. Kinases phosphorylate protein substrates in a highly regulated manner and are promiscuous. Understanding kinase-substrate specificity has been challenging and there is a need for new chemical tools. To this end we developed -phosphate modified ATP photocrosslinking analogs ATP-ArN3 and ATP-BP, that crosslink substrate and kinase in a phosphorylation dependent manner. We have successfully demonstrated that ATP-ArN3 and ATP-BP can be used with natural kinase and substrates using cell lysates in vitro. We used our approach to identify novel kinases of …

Computational Approaches To Anti-Toxin Therapies And Biomarker Identification, Rebecca Jane Swett

Wayne State University Dissertations

This work describes the fundamental study of two bacterial toxins with computational methods, the rational design of a potent inhibitor using molecular dynamics, as well as the development of two bioinformatic methods for mining genomic data.

Clostridium difficile is an opportunistic bacillus which produces two large glucosylating toxins. These toxins, TcdA and TcdB cause severe intestinal damage. As Clostridium difficile harbors considerable antibiotic resistance, one treatment strategy is to prevent the tissue damage that the toxins cause. The catalytic glucosyltransferase domain of TcdA and TcdB was studied using molecular dynamics in the presence of both a protein-protein binding partner and …

"Fine-Tuning" Of Ribosomal Structure And Functions By Pseudouridylation And Rna-Protein Interactions, Jun Jiang

Wayne State University Dissertations

ABSTRACT

"Fine-tuning" of ribosomal structure and functions by pseudouridylation and RNA-protein interactions

by

JUN JIANG

AUGUST 2012

Advisor: Prof. John SantaLucia Jr.

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

Ribosomal structure and functions appear to be "fine-tuned" by pseudouridylation and RNA-protein interactions. Pseudouridylation may promote base stacking interactions by mediating the base stacking between residues on both sides. In the RNA duplex region, this enhanced stacking interaction contributes to stabilization of duplex folding. In the loop region, enhanced stacking in one structural motif may destabilize the conformation of adjacent structural residues. This hypothesis is supported by both UV-melting experiments, where …

The Structural Requirements Of Histone Deacetylase (Hdac) Inhibitors: Suberoylanilide Hydroxamic Acid (Saha) Analogues Modified At C3, C6, And C7 Positions Enhance Selectivity, Sun Ea Choi

Wayne State University Dissertations

Histone deacetylase (HDAC) proteins are targets for drug design towards the treatment of cancers since overexpression of HDAC is linked to cancer. Several HDAC inhibitors, including the FDA approved drug suberoylanilide hydroxamic acid (SAHA, Vorinostat), have cleared clinical trials and emerged as anti-cancer drugs. However, SAHA inhibits all of the 11 metal ion-dependent HDAC proteins. Therefore, we synthesized several libraries of small molecule HDAC inhibitors based on SAHA to help understand the structural requirements of inhibitory potency and isoform selectivity.

In previous work, SAHA analogues functionalized at the C2 position (C2-SAHA analogues) near the metal binding hydroxamic acid displayed decreased …

Development Of A Cargo Delivery System And Inhibition Studies Focused On Clostridium Difficile Toxin A, Stephanie Marie Kern

Wayne State University Dissertations

Virulence factors of pathogenic bacteria are to be blamed for life-threatening infections such as diphtheria, anthrax, botulism, and tentanus. In the case of enzymatic exotoxins, disease arises from cytotoxic proteins, and cytotoxicity is acheived only after cell entry. This intrinsic mechanism for cell entry is intriguing from research and medical views. Along with a review on existing cargo delivery systems utilizing protein toxins and the usefulness of such a system, here is described the first reported Clostridium difficile toxin A fusion protein, luciferase-TcdA, and evidence of the successful transport of an active enzyme, luciferase, into the cytosol of vero cells. …

Exploring Conformational Variability Of An Rna Domain In The Ribosome: From Structure And Function To Potential Antibiotic Targeting, Yogo Sakakibara

Wayne State University Dissertations

RNA in nature is modified at many specific sites to order to gain extra functions or to expand the genetic code. One of such RNAs is ribosomal RNA (rRNA), which contains several modified bases, particularly around the functionally significant sites. We have focused on understanding the influences of modified base on RNA structure and function by employing helix 69 (H69), which is a good region to evaluate the roles of modified bases since it contains three pseudouridines in the loop region and exists at the core of the ribosome.

Previous model studies using small hairpin H69 showed the conformational differences …

Drug Resistance Mechanisms And Drug Design Strategies For Human Immunodeficiency Virus And Hepatitis C Virus Proteases, Yong Wang

Wayne State University Dissertations

The antiviral drug development has improved steadily to treat the infections of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) which represent heavy public health burdens. The viral protease plays an indispensable role in viral maturation and therefore becomes one of the most important targets for drug design. Nine HIV-1 protease inhibitors and two HCV protease inhibitors have been developed and approved by the U.S. Food and Drug Administration. However, mutations in the protease decrease reduce the efficacy the drugs. In this study, the enzyme assays indicate that darunavir and tipranavir exhibit the most potent inhibition against the multi-drug …