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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 …

Biochemical, Structural, And Drug Design Studies Of Multi-Drug Resistant Hiv-1 Therapeutic Targets, Tamaria Grace Dewdney

Wayne State University Dissertations

Protein point mutations acquired as a mechanism of survival against therapeutics cause structural changes that effect protein function and inhibitor binding. This work investigates the structural mechanisms that lead to multi-drug resistance to HIV-1 protease and integrase inhibitors.

Proper proteolytic processing of the HIV-1 Gag/Pol polyprotein is required for HIV infection and viral replication. This feature has made HIV-1 protease an attractive target for antiretroviral drug design for the treatment of HIV-1 infected patients, thus the development of drug resistance has arisen as a major therapeutic and drug design challenge. To understand the molecular mechanisms leading to drug resistance we …

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 …

Enzymatic Characterization Of The Ammonia Tunnel In Helicobacter Pylori Asp-Trnaasn/Glu-Trnagln Amidotransferase, Liangjun Zhao

Wayne State University Dissertations

The Helicobacter pylori (H. pylori) Asp-tRNA^Asn/Glu-tRNA^Gln amidotransferase (AdT) plays important roles in indirect aminoacylation and translational fidelity; however, its inter-domain communication and ammonia delivery mechanisms are not well understood. In the present study, we investigated the three activities of H. pylori AdT (glutaminase, kinase and transamidase) and used these reactions as probes to examine the inter-domain communication and ammonia delivery mechanisms between this enzyme's two isolated active sites. We adapted and optimized an assay to kinetically characterize a series of mutations at conserved positions throughout the putative AdT ammonia tunnel. The kinase assay enabled us …

Histone Deacetylase 1: Mutagenesis And Small Molecule Studies, Magdalene Wambua

Wayne State University Dissertations

Histone deacetylase 1 (HDAC1) has been linked to cell growth and cell cycle regulation, which makes it a widely recognized target for anticancer drugs. The 14 Å channel of Class 1 HDAC isotypes has long being hypothesized to be the exit cavity for acetate following deacetylation. The amino acids lining this cavity are very similar among the HDAC isoforms, suggesting the role of the cavity is relevant to all HDACs proteins Importanly, HDAC1 selective inhibitors designed to fit the 14Å channel have been designed. To understand the importance of the 14Å channel to HDAC1 activity, we used an alanine scan …

The Role Of Cardiolipin In Iron Homeostasis And Glutathione Metabolism, Vinay A. Patil

Wayne State University Dissertations

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. Inside the mitochondria, CL is a critical target of mitochondrial generated reactive oxygen species (ROS) and regulates signaling events related to apoptosis and aging. CL deficiency causes perturbation of signaling pathways outside the mitochondria, including the PKC-Slt2 cell integrity pathway and the high osmolarity glycerol (HOG) pathway, and is a key player in the cross-talk between the mitochondria and the vacuole. The importance of CL in human health is underscored by the observation that perturbation of CL …

"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. …

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 …

Single Molecule Studies Of Rna-Target Interactions, Sharla Leann Wood

Wayne State University Dissertations

We have used FRET, single molecule spectroscopy, and several other biophysical techniques to study how the folding of RNA allows it to perform its various functions by recognizing and binding a target ligand. We have shown that the c-di-GMP riboswitch undergoes a large, global conformational change upon binding of the ligand. The folding dynamics of the c-di-GMP riboswitch upon Mg2+ binding help to pre-organize the aptamer for efficient ligand binding and ultimately efficient gene expression. We have also investigated two instances, a fluorophore-binding aptamer and molecular beacon, where the folding of RNA can be used to detect an analyte. We …

Understanding The Gender-Based Mechanism Of Mso In Als Mice: A Metabolic Characterization Of The Sod1-G93a Mouse Model, Monica Ann Bame

Wayne State University Dissertations

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by motor neuron death and a corresponding loss of neuromuscular connections resulting in muscle atrophy. Patients become paralyzed shortly after symptom onset and typically die within one to five years of pulmonary complications. ALS is a relatively rare disease, with an overall incidence of approximately 2 in 100,000 people per year and a prevalence of about 5 in 100,000 people. It is typically associated with increasing age and has a slight male prevalence, with a male to female ratio of approximately 3:2. ALS is classified as either familial (the less …

Transfer Rna Dynamics And Transfer-Messenger Rna Accommodation In Bacterial Ribosomes At The Single-Molecule Level, May Daher Farhat

Wayne State University Dissertations

Single-molecule spectroscopy, protein-induced fluorescence enhancement (PIFE), fluorescence resonance energy transfer (FRET), and several biochemical tools were applied to study transfer RNA (tRNA) dynamics and transfer-messenger RNA (tmRNA) accommodation inside ribosomes. In the first project of this thesis work, structural characterization of the tRNA-like domain of tmRNA (TLD) in complex with SmpB protein was carried out, and the results reveal no change in the global conformation or the flexibility of the TLD upon SmpB binding. In contrast, magnesium ions induce a compaction of the TLD structure, suggesting that flexibility in the H2a stem of TLD may allow different conformations of tmRNA, …

Development Of An In-Cell Förster Resonance Energy Transfer Technique To Study Protein Structure Inside Living Cells, Victoria Lynn Murray

Wayne State University Dissertations

The goal of my thesis is to develop an in-cell fluorescence technique that allows for measurement of the distances between fluorescence acceptors and donors within a protein or between two proteins inside the correct intracellular compartment of living cells. The successful achievement of this goal will allow us to obtain high-resolution structural information from a protein, one key step towards high-resolution structural biology of proteins inside the living cell.

To achieve this goal, we will apply the fluorescence resonance energy transfer (FRET) technique to the specifically labeled proteins inside the cells. Our rationale is to specifically label the protein(s) of …

Modes And Mechanisms Of Hfq Mediated Stress Regulation In Bacteria, Nilshad Nilam Salim

Wayne State University Dissertations

To survive bacteria must be able to respond to its ever-changing environmental conditions. sRNAs have been implicated in a variety of stress-response pathways that help bacterial systems modulate gene expression. The RNA binding protein Hfq facilities this process by, helping sRNA to base pair with its target mRNAs to initiate gene regulation. A common feature of Hfq-mediated gene regulation is the network-based organization where a single sRNA can control multiple messages to promote integrated response to stress. Current mechanistic models that are present to describe Hfq functions cannot explain the complexity at which Hfq performs gene regulation. In this work …

Prevalence And Physiological Significance Of Gene Looping In Saccharomyces Cerevisiae, Banupriya Mukundan

Wayne State University Dissertations

My Ph.D. dissertation work is focused on studying the role of promoter-bound transcription initiation factors involved in gene looping. In this study we showed that the RNAP II subunit Rpb4 has a significant effect on termination of transcription. Gene looping is disrupted in the absence of Rpb4. Rpb4 shows a strong physical interaction with the Mediator subunit Srb5. Mediator subunit Srb5 crosslinked to the 5' and 3' ends of INO1 and CHA1 genes and is required for proper termination of transcription of these genes. Srb5 affected termination of transcription through its interaction with the CF1 complex. Srb5 interaction with the …

Genetic And Biochemical Studies Of Human Apobec Family Of Proteins, Priyanga Wijesinghe

Wayne State University Dissertations

The AID/APOBEC family of proteins in higher vertebrates converts cytosines in DNA or RNA into uracil. These proteins have essential roles in either innate immunity or adaptive immunity. Recently, AID has also been implicated in DNA demethylation in the context of early embryogenesis in mammals. This is partly based on the reported ability of AID to deaminate 5-methyl cytosine to thymine (5mC to T). I reexamined this proposed new role of AID (5mC deamination) with two members of the APOBEC family in a novel Escherichia coli based genetic system. My results confirmed that while all three enzymes are strong cytosine …

Use Of Methionine Sulfoximine To Dissect The Role Of Glutamine Synthetase And Glutamine In Progression Of Acute Liver Failure, Amruta Anil Jambekar

Wayne State University Dissertations

Methionine sulfoximine (MSO) is a modified amino acid and a well characterized irreversible inhibitor of glutamine synthetase (GS) enzyme. Glutamine is synthesized by GS enzyme and it is the most abundant amino acid in the body. Glutamine is required by immune cells for generation, propagation and maintenance of an immune response.

To induce acute liver failure (ALF) in mice, animals were given intraperitoneal injections of E. coli lipopolysaccharides (LPS) and sugar D-galactosamine (D-GalN). When these animals were pretreated with MSO, 80% of the animals were completely rescued from liver failure. Moreover, when we characterized the immune response generated during ALF …

The Role Of Cad,Flash And Fam129b In Cancer Cell Survial And Apoptosis, Song Chen

Wayne State University Dissertations

Apoptosis is a normal process in the human body. However, apoptosis is desregulated in cancer cells. Most cancer cells gain resistance to apoptosis, leading to uncontrolled proliferation. In this dissertation, we identified three proteins, associated with apoptosis pathway. 1) CAD, a large multifunctional complex that is invariably elevated in tumor cells, 2) FLASH, a large protein with multiple growth related functions and 3) FAM129B. We demonstrate that CAD could interact with FLASH by using yeast two hybrid, co-immunopreciptation and fluorescence microscopy. In addition, functional analysis using siRNA technology further indicated that CAD could co-operate with FLASH and play roles in …

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 …

Cardiac Calsequestrin Phosphorylation And Trafficking In The Mammalian Cardiomyocyte, Timothy Mcfarland

Wayne State University Dissertations

Cardiac CSQ (CSQ2) is a multifaceted protein, capable of binding significant quantities of Ca2+ and altering ryanodine receptor activity at the junctional sarcoplasmic reticulum (SR). Little is known about the trafficking of CSQ2 from its unknown site of biosynthesis, which appears to be of importance as its structure changes in a trafficking-dependent manner in various types of heart failure. Through the use of multiple antibodies specific to classic rough ER markers, and with the creation of CSQ-DsRed tetramer fusion protein, we were able to establish a juxtanuclear localization of rough ER in cardiomyocytes. Using fluorescence confocal microscopy, the translocon complex …

Exploring Potential Drug Target Sites In The Ribosome Using Cisplatin And Its Analogues, Keshab Rijal

Wayne State University Dissertations

Cis-diamminodichloridoplatinum (II), cisplatin, is an antitumor drug that has been used to treat several types of cancers. The reaction of cisplatin with DNA has been studied and discussed extensively in the literature; however, the effects of cisplatin on RNA function are poorly understood. In this thesis, two aspects of cisplatin, its preferred sites of interaction with RNA and its use as a chemical probe to gain accessibility information, were explored.

To understand the site-selectivity of cisplatin with RNA, model RNA constructs and full-length 16S rRNA were employed. The binding studies revealed a cisplatin preference for guanosine-rich sequences. Primer extensions in …

Molecular Details Of The Mitochondrial Iron Sulfur Cluster Assembly Pathway, Swati Rawat

Wayne State University Dissertations

MOLECULAR DETAILS OF THE MITOCHONDRIAL IRON SULFUR CLUSTER ASSEMBLY PATHWAY

Iron-sulfur clusters are an important class of prosthetic group involved in electron transfer, enzyme catalysis, and regulation of gene expression. Their biosynthesis requires complex machinery located within the mitochondrion since free iron and sulfide are extremely toxic to the cell. Defects in this pathway results in several diseases such as Friedreich's Ataxia (FRDA), Sideroblastic Anemia and ISCU Myopathy. Therefore molecular details of the biogenesis pathway will provide deep insight in the pathway and treatment options for these diseases. FRDA is caused by deficiency of a single protein called as `Frataxin'. …

Characterization Of Splicing Mechanisms By Single-Molecule Fluorescence, Krishanthi Sanjeewani Karunatilaka

Wayne State University Dissertations

Group II introns rank amongst the largest self-splicing ribozymes found in bacteria and organellar genomes of various eukaryotes. Despite the diversity in primary sequences, group II introns posses highly conserved secondary structures consisting of six domains (D1-D6). To perform its function, the large multidomain group II intron RNA must adopt the correctly folded structure. As a result, in vitro splicing of these introns requires high ionic strength and elevated temperatures. In vivo, this process is mainly assisted by protein cofactors. However, the exact mechanism of protein-mediated splicing of group II intron RNA is still not known.

In order to …

Effects Of Dietary Fat Saturation On Lipoprotein Metabolism In Rodents And Humans, Deepinder Kaur

Wayne State University Dissertations

Consumption of trans-fatty acids (tFA) and saturated fatty acids (SFA) have been associated with higher incidence of coronary heart disease. Experimental and observational data suggests that consumption of PHVO containing tFA, like SFA leads to increased total cholesterol and LDL-cholesterol (LDL-C) but compared to tFA, SFA raises HDL-cholesterol (HDL-C). An attempt to eliminate PHVO from the food supply has resulted in the need to find alternatives which despite their higher dietary fat saturation as compared to naturally occurring vegetable oils, may be preferable to PHVO. However, amongst SFA, the effects on plasma lipoproteins are variable. Thus, we hypothesized that specific …

A Novel In Vivo Protein Refolding Technique, Yuefei Huang

Wayne State University Dissertations

Proteins perform their functions in their native folded states and misfolding of proteins may cause severe diseases, including Alzheimer's disease, Parkinson's disease, prion disease and diabetes. Understanding protein folding is important for us to engineer proteins to treat these diseases. For protein therapeutics, large quantities of properly folded and functional proteins are required. The current technology produces recombinant proteins using either eukaryotic or prokaryotic expression system, both of them have major problems that prevent production of large quantities of properly folded and functional human proteins for protein therapeutics.

Although the eukaryotic cells have comprehensive folding machinery that contains chaperones and …

Destruction Of Biological Tetrapyrrole Macrocycles By Hypochlorous Acid And Its Scavenging By Lycopene, Dhiman Maitra

Wayne State University Dissertations

Hypochlorous acid (HOCl) is a potent oxidant generated by the hemoprotein myeloperoxidase. Although HOCl plays an important role in the innate immune response,sustained high levels of HOCl has been implicated to play a harmful role. In several pathological conditions such as atherosclerosis, endometriosis and sickle cell disease where HOCl is elevated there are reports of significant free iron accumulation. Free iron is toxic since it can lead to the generation of other secondary free radicals such as hydroxyl radical by Fenton reaction. The exact source and mechanism by which the free iron is generated is not clearly understood. This work …

Method Development And Applications To Screening And Characterization Of Rrna-Targeting Small Molecules, Papa Nii Asare-Okai

Wayne State University Dissertations

A series of single ring aminoglycoside analogues was tested for binding to a model RNA representing the A site using electrospray ionization mass spectrometry (ESI-MS). Several of the synthetic analogues with low molecular weights were found to bind to the RNA with affinities comparable to the parental aminoglycoside neamine, with apparent dissociation constants in the low micromolar range. Salt dependence of the affinity constants for the single ring analogues revealed a predominantly electrostatic binding mode. Footprinting experiments revealed that one of the compounds (DHR23) has a similar binding site as the antibiotic paromomycin. DMS chemical probing results also suggest that …

Nmr Solution Structures Of Two Hairpins Of E. Coli 16s Rrna: The Effects Of Mutations And Chemical Modifications On Structure And Function Of Rrna, Yu Liu

Wayne State University Dissertations

The structures of two functional mutants, the UC (G690U, U697C) and the QM mutants (G690A, G693C, A695C, U697A) of the 690 hairpin of E. coli 16S ribosomal RNA were determined by NMR. The UC mutant and the QM mutant with high biological function are able to fold into structures that are isomorphous with the wild-type 690 hairpin sequence. The structural comparisons among the functional mutants and the wild-type provides structural validation for previously identified specific functional groups that are crucial for maintaining function of the 690 hairpin. The key groups for maintaining the structure and function of the 690 loop …