Biochemistry Commons^™

Functional Characterization Of Threonine 49 Phosphorylation Of Cytochrome C, Antoine Khobeir

Wayne State University Theses

Cytochrome c (Cytc) is a pivotal multifunctional mitochondrial protein that serves as a single electron carrier between complexes III and IV of the electron transport chain. It has important roles in both cellular respiration and apoptosis. The novel Thr49 (T49) phosphorylation of Cytc likely affects mitochondrial respiration, membrane potential, ROS production, ATP production, and apoptosis. Based on the functional characterization of previously mapped phosphorylation sites (Tyr97, Tyr48, Thr28, Ser47, Thr58) of the lab, we hypothesize that T49 phosphorylation will lead to controlled respiration, optimal intermediate mitochondrial membrane potential, lower ROS production, and inhibition of apoptosis compared to unphosphorylated Cytc. Here …

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, …

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 …

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 …

Fam129b Phosphorylation And Its Effect On Membrane Localization In Confluent Hela Cells, Lakshmi Thompil Somasekharan

Wayne State University Theses

Phosphorylation and de-phosphorylation of many proteins is a key regulator of cellular life. It maintains cellular activity through an array of signaling pathways like cell division, proliferation and growth. However, Overexpression or mutations by the constitutive activation of phosphorylation machinery disrupt the balance in the cell, driving the inappropriate activation or deactivation of the cellular processes it controls. FAM129B is a protein whose activity is partly maintained by phosphorylation and dephosphorylation at the six serine residues at the C-terminal. FAM129B is expressed highly in many forms of cancer and its main function is to suppress apoptosis and enhances cancer cell …

The Effect Of Acetylation Of Cytochrome C On Its Functions In Prostate Cancer, Viktoriia Bazylianska

Wayne State University Theses

Prostate cancer is the second leading cause of cancer death among men in America. The progression of cancer goes along with the Warburg effect, a metabolic switch from depending primarily on mitochondrial respiration to glycolysis. In addition, cancer cells manage to evade apoptosis. Cell signaling, via posttranslational modifications (PTMs), is one of the most important means of regulation, and most commonly dysregulated in cancer. In prostate cancer, androgen signaling plays a crucial role in driving cell proliferation.

Mammalian Cytochrome c (Cytc) is a multifunctional protein involved in cellular life and death decision. It is an essential component of the electron …

Elucidating Structure, Function, And Small Molecular Interactions Of Human Immunodeficiency Virus And Chikungunya Virus, Kristin Nicole Slater

Wayne State University Theses

Abstract HIV-1:

Human immunodeficiency virus-1 (HIV-1) is a widespread, incurable retrovirus known to cause

immunodeficiency and a shortened life span. Despite successful treatment methods, HIV-1

frequently mutates, resulting in antiviral resistance. Many therapies target the HIV-1 protease

(PR), which is responsible for cleaving the viral polyprotein essential for its life cycle. HIV-1 PR

often evades treatment by way of mutations and less commonly through residue insertions. We

have identified a clinical isolate with a five residue insertion between residues 28 and 29.

Through molecular dynamics simulations we analyzed the protease protein structure and

determined that the residue insertion created a …

Expression, Purification And Characterization Of Lysine Methyltransferase Smyd5, Wen Xue

Wayne State University Theses

Methylation of histones and non-histone proteins play vital roles in numerous cellular processes including gene expression regulation and DNA damage response. The identifications of methyltransferase SMYD protein family are not well characterized. SMYD5 is a unique and critical member of SMYD family involved in immune response, stem cell renew, hematopoiesis regulation and cancer metastasis. Understanding its function and structure is monumental to human disease. With the achievement of SMYD5 expression and purification, the association between SMYD5 and its substrate histone H4 has been investigated. While possessing a poly-E tail instead of the TPR domain included in other members, SMYD5 is …

Studies Of Sumoylation In Regulating Mif Stability And Rangap1 Nucleo-Cytoplasmic Shuttling In Controlling Its Sumo Modification, Progga Sen

Wayne State University Dissertations

SUMOylation is an essential post-translational modification that regulates a variety of critical cellular pathways ranging from nuclear transport to protein stability. Accumulating lines of evidence have shown that a perturbation of the SUMOylation pathway is associated with human diseases, especially various types of cancer. Our recent proteomic studies revealed a drastic increase in levels of SUMO2/3 modification on the proinflammatory cytokine MIF in the metastatic breast cancer cell line compared to the non-metastatic control cell line. Interestingly, the increase in levels of both MIF and global SUMO-2/3 modification in the metastatic cells are positively correlated to that of unmodified MIF …

Development Of A Novel Class Of Chemicals For Labeling Abasic Sites In Cellular Dna And Killing Cancer Cells, Shanqiao Wei

Wayne State University Dissertations

Abasic (AP) sites are the most common type of lesions in DNA. Numerous endogenous and exogenous agents and cellular processes can induce the formation of AP sites in DNA. If left unrepaired, the deleterious AP sites cause mutagenesis and cytotoxicity. Methoxyamine is known to react with AP sites and block base excision repair. Another alkoxyamine, aldehyde-reactive probe (ARP) tags AP sites with a biotin and has been widely used to quantify these sites. In this study, I have combined both these abilities into one alkoxyamine, AA3, which reacts toward AP sites with better reactivity than ARP at physiological pH. Additionally, …

Insights Into De Novo Fes-Cluster Biogenesis Via The Eukaryotic Fes-Cluster (Isc) Pathway In Vitro, Stephen Paul Dzul

Wayne State University Dissertations

Fe-S clusters are iron-containing cofactors utilized by numerous proteins within several biological pathways essential to life. In eukaryotes, the primary pathway for Fe-S cluster production is the iron-sulfur cluster (ISC) pathway. The eukaryotic ISC pathway, localized primarily within the mitochondria, has been best characterized within Saccharomyces cerevisiae. In yeast, de novo Fe-S cluster formation is accomplished through coordinated assembly of the substrates iron and sulfur on the primary scaffold assembly protein “Isu1”. The sulfur used for cluster assembly is provided by the cysteine desulfurase “Nfs1”, a protein that works in union with its accessory protein “Isd11”. Frataxin “Yfh1” helps direct …

A Critical Role Of Cxcr2 Pdz Motif-Mediated Interactions In Endothelial Progenitor Cell Homing And Angiogenesis, Yuning Hou

Wayne State University Dissertations

Bone marrow-derived endothelial progenitor cells (EPCs) participate in postnatal vascularization in response to growth factors, cytokines, and chemokines. Chemokine receptor CXCR2 and its cognate ligands are reported to mediate EPC recruitment and angiogenesis. CXCR2 possesses a consensus PSD-95/DlgA/ZO-1 (PDZ) motif at its carboxyl terminus. The PDZ motif has been reported to regulate cellular signaling and functions. Here we investigated the potential role of the PDZ motif in CXCR2-mediated EPC motility and angiogenesis. We have found that introducing exogenous CXCR2 C-terminus significantly attenuated in vitro EPC migration and angiogenic activities in response to CXCR2 ligands, as well as in vivo EPC …

Investigation Of Mutations In Nuclear Genes That Affect The Atp Synthase, Russell Dsouza

Wayne State University Dissertations

The F1 domain is the catalytic subunit of the mitochondrial ATP synthase. Studies with respiratory-deficient yeast identified ATP1 and ATP2 as nuclear genes encoding the alpha and beta subunits, respectively, of the mitochondrial F1-ATPase. The mutations in the atp1 and atp2 genes were cloned and sequenced, and they appear to affect the ATP synthase. Most yeast strains with mutations in the or the subunit primarily show an F1 assembly defective phenotype. This feature is similar to the assembly-defective mutants missing the chaperones required for assembly of the F1 oligomer or either the alpha/beta subunits.

Some of the atp2 and atp1 …

Novel Regulatory Mechanisms Of Inositol Biosynthesis In Saccharomyces Cerevisiae And Mammalian Cells, And Implications For The Mechanism Underlying Vpa-Induced Glucose 6-Phosphate Depletion, Wenxi Yu

Wayne State University Dissertations

Myo-inositol is the precursor of all inositol containing molecules, including inositol phosphates, phosphoinositides and glycosylphosphatidylinositols, which are signaling molecules involved in many critical cellular functions. Perturbation of inositol metabolism has been linked to neurological disorders. Although several widely-used anticonvulsants and mood-stabilizing drugs have been shown to exert inositol depletion effects, the mechanisms of action of the drugs and the role of inositol in these diseases are not understood. Elucidation of the molecular control of inositol synthesis will shed light on the pathologies of inositol related illnesses.

In Saccharomyces cerevisiae, deletion of the four glycogen synthase kinase-3 genes, MCK1, MRK1, MDS1, …

Contribution Of Cx3cr1/Fractalkine (Cx3cl1) Axis To The Progression Of Non-Small Cell Lung Cancer (Nsclc), Rhonda Prince

Wayne State University Theses

A thesis is presented on the characterization of the contribution of the chemokine axis, CX_3CR1/Fractalkine, to the progression of Non-Small Cell Lung Cancer (NSCLC). Cancer is the second leading cause of death in the United States, and Lung cancer is one of the four leading causes of the disease. Carcinoma cells, unlike their normal counterparts, gain the ability to express chemokine receptors. Understanding if/how this gain of expression contributes to the progression of cancer could provide a lucrative therapeutic target. Goals of the study include investing if the NSCLC cell line A549, an adenocarcinoma cell line, express the chemokine receptor …

Characterization Of The Yeast Cysteine Desulfurase Complex Within The Mitochondrial Fe-S Cluster Biogenesis, Dulmini Pabasara Barupala

Wayne State University Dissertations

Disrupted iron homeostasis within the human body materializes as various disorders. Pathophysiology of many of them relates to iron induced oxidative damage to key cellular components caused by iron accumulation within the tissues. Pertaining to the growing occurrence, cost of patient care and devastating burden associated with these diseases, the call for understanding the role of iron homeostasis within these disorders becomes inevitable. Being an abundant iron containing cofactor, the role of Fe-S clusters in cellular iron homeostasis is indisputable in the case of Friedreich’s ataxia, a disease caused by a deficiency in the protein frataxin that is indispensable during …

Cardiolipin Is Required For Optimal Acetyl-Coa Metabolism, Vaishnavi Raja

Wayne State University Dissertations

The phospholipid cardiolipin (CL) is crucial for many cellular functions and signaling pathways, both inside and outside of mitochondria. My thesis focuses on the role of CL in energy metabolism. Many reactions of electron transport and oxidative phosphorylation, the transport of metabolites needed for these processes, and the stabilization of electron transport chain supercomplexes, require CL. Recent studies indicate that CL is required for the synthesis of iron-sulfur (Fe-S) co-factors, which are essential for numerous metabolic pathways. Activation of carnitine-acetylcarnitine translocase, which transports acetyl-CoA into the mitochondria, is CL dependent. The presence of substantial amounts of CL in the peroxisomal …

Analyzing The Interactions Of Kdm5/Lid And Sin3 In Drosophila Melanogaster, Ambikai Gajan

Wayne State University Dissertations

SIN3, the scaffold protein of a histone modifying complex is conserved from yeast to mammals. Drosophila SIN3 associates with both a histone deactylase RPD3 and a histone demethylase dKDM5/LID. Immunopurification of dKDM5/LID verifies a previously observed interaction with SIN3 and RPD3. Furthermore, deficiency of dKDM5/LID phenocopies deficiency of SIN3 in many cellular and developmental processes. Knockdown of both Sin3A and lid hinder cell proliferation in Drosophila cultured cells and developing flies. Knockdown of these genes also results in a curved wing phenotype implicating a role in wing development. Analysis of underlying gene expression changes upon decreased expression of SIN3, dKDM5/LID …

In Vitro Characterization Of Serine 47 Phosphorylated Cytochrome C, Ashwathy Mary Varughese

Wayne State University Theses

Cytochrome c is a 12.4kDa ubiquitously expressed protein known to be involved in many physiological processes of the cell such as respiration and apoptosis. The goal of our lab is to increase our knowledge of the regulation of cytochrome c in these opposite activities, and our working model posits that cytochrome c is decisively regulated by phosphorylation. When phosphorylated, cytochrome c leads to an "optimal" functioning in the electron transport chain by lowering electron flux, preventing harmful high mitochondrial membrane potentials and thus ROS production under healthy conditions. However, under cellular stress cytochrome c might be dephosphorylated favoring high mitochondrial …

Investigating The Metal Binding Affinity Of Bacterial Zinc Transporters: Zupt And Truncated Znta (Znta-Pina), Fei Nie

Wayne State University Theses

Zinc is essential trace elements for all living organisms and serves as cofactor and structural cofactors for more than 300 enzymes. ZupT is one of the members of ZIP super family of transporter proteins, which were identified as iron and zinc transporter in Eukaryotes at first. However, recent evidence showed that some of members can also transport other metal ions, such as manganese or cadmium, and ZIP proteins also exist in bacteria. ZupT is responsible for zinc uptake in E. coli; however, its full metal selectivity is not yet characterized. In addition, the mechanism of transport is also unknown. Therefore, …

Characterizing Cyclin J By Identifying Conserved Protein-Protein Interactions, Phillip Jacob Selman

Wayne State University Theses

Cyclins are proteins that bind to Cyclin-dependent kinases, or Cdks, through a conserved domain called the Cyclin Box. Many Cyclins regulate the cell cycle. A few Cyclins impact cellular processes outside of the cell cycle. Also, a few Cyclins have poorly understood functions.

Cyclin J is a member of the Cyclin superfamily of proteins. Cyclin J is conserved among all metazoans, but is presently not well understood. All the research done on Cyclin J has been done in Drosophila.

Its mRNA is present in the early embryo, then disappears, only to reappear in adult females. When probing protein extracts with …

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 …

Optimization Of Labeling Techniques; Determination Of Best Parameter For Olfactory Mucosal Progenitor Cell Delivery And Study Of Effects Of Methylene Blue And Polyethylene Glycol In An Animal Model Of Spinal Cord Injury, Kiran Kumar Rokkappanavar

Wayne State University Theses

In the United States, the incidence of TSCI is about 40 per million persons per year, with approximately 250,000 living survivors of TSCI in the United States in July 2005. A number of human clinical trials (85) are ongoing using stem cells to evaluate the methods to reduce the injury following TBI. Among the stem cells, olfactory mucosal progenitor cells have several advantages with respect to ease of obtaining, fate of cells, etc. Our study was intended to optimize the labeling technique and to determine the best parameter to deliver the stem cells to the site of injury. We found …

Investigation Of Bacterial Rna-Directed Dna Methylation Via Dcm And Hfq, Dandan Li

Wayne State University Theses

Bacterial small RNAs and the RNA chaperone Hfq play crucial roles in post-transcriptional gene regulation, often as parts of stress-response pathways, but little is known about their roles in regulation of gene transcription. A recent report showed that changes in methylation patterns caused by DNA cytosine methyltransferase (Dcm) were linked to gene regulation occurring during the transition to stationary phase. Here, we show that Dcm involves in the stress responses under nutrient starvation and cold stress. Dcm and Hfq together mediate gene expression under cold stress. Hfq promotes Dcm-catalyzed cytosine methylation at specific sites near the rpoS promoter, which is …

Cysteine Desulfurase And Isd11: A Drosophila Model, Rahul Ravindran Nair

Wayne State University Theses

ABSTRACT

Cysteine desulfurase and Isd11: A Drosophila model

Iron-sulfur clusters are cofactors with evolutionary origins that date back to the pre-biotic world. Ever since life originated, these cofactors have intermingled with proteins to play vital roles in sustaining life. My research focuses on one such protein, the cysteine desulfurase (Nfs) that has the PLP cofactor incorporated in its active site and avails of the catalytic property of PLP to provide sulphur for Iron-sulfur cluster biogenesis and assembly in a cell. Interestingly, in a eukaryotic cell, despite the versatility of PLP, cysteine desulfurase's role as a "sulphur-extractor" is incomplete without another …

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 …

Disrupting Cxcr2 Macromolecular Complex Pdz-Domain Interactions During Inflammatory Chemotaxis, Marcello Castelvetere

Wayne State University Theses

Neutrophils are the body's first responders to inflammation, being the most abundant white blood cell type in circulation and they quickly initiate an immune response through chemokine signaling. Inflammatory chemokines signal via their receptor CXCR2, which initiates an inflammatory response, recruiting leukocytes to sites of inflammation. Chemokine signaling is important for proper host protection, yet uncontrolled activity is responsible for a variety of pathological conditions: including rheumatoid arthritis, ischemia-reperfusion injury, arteriosclerosis, multiple sclerosis, psoriasis, inflammatory bowel disease, and allergic reactions.

In this report I show a CXCR2 macromolecular signaling complex exists in neutrophils, containing NHERF1 and PLCβ2. I also demonstrate …

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 …