Molecular Biology Commons^™

Post-Translational Control Of Retinoblastoma Protein Phosphorylation, Paul M. Stafford

Electronic Thesis and Dissertation Repository

The retinoblastoma tumor suppressor protein (pRB) functions through multiple mechanisms to serve as a tumor suppressor. pRB has been well characterized to be inactivated through phosphorylation by CDKs. pRB dephosphorylation and activation is a much less characterized aspect of pRB function. In this thesis, I detail work to study the post translational control of pRB phosphorylation. Here I present work detailing efforts to generate a gene targeted mouse which disrupts PP1 binding to the C-terminus of pRB, allowing for detailed study of the mechanisms of pRB dephosphorylation. This work also details an examination of acetylation in the C-terminus of pRB, …

Regulation Of Saga By The N-Terminus Of Spt7 In Saccharomyces Cerevisiae, Dominik Dobransky

Electronic Thesis and Dissertation Repository

Spt7 is a 1,332 residue protein critical for maintaining structural integrity of the SAGA complex. I demonstrated that the extreme N-terminus of Spt7 plays an important role in SAGA function. Deletion of the first 73 (Spt7_73-1332) and 121 (Spt7_121-1332) N- terminal residues resulted in slow growth, decreased transcriptional activation at PHO5 and INO1, and a partial decrease in acetylation at lysine 18 of histone H3 at PHO5. The Spt7_121-1332 mutant did not affect Spt7’s association with Gcn5 or Tra1, or its localization within the cell. Mutation of the first four positively charged residues …

The Role Of The Pre-Sensor 1 Β Hairpin In Minichromosome Maintenance 2-7 Function, Simon K. W. Lam

Electronic Thesis and Dissertation Repository

The pre-sensor 1 (PS1) hairpin is found in helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding. To determine whether the PS1 b hairpin is required in the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the PS1 hairpin in each of the S. cerevisiae Mcm subunits to alanine. Only the PS1 hairpin of Mcm3 was essential for viability, while mutation of the PS1 hairpin in the remaining Mcm subunits resulted in minimal phenotypes, with the exception …

Exploring The Structure And Biochemistry Of Oxidation-Mediated Inhibitation Of The Peptidyl-Prolyl Isomerase Pin1, Brendan T. Innes

Electronic Thesis and Dissertation Repository

Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase that has been shown to be neuroprotective in aging-related neurodegenerative diseases such as Alzheimer's disease (AD). However, it is not active in AD brain, and a recent proteomic screen of Mild Cognitive Impairment (MCI) brain samples revealed that Pin1 is oxidized in the brains of these pre-AD patients. This suggests that this oxidation may be the cause of the loss of the neuroprotective Pin1 function in AD. The Pin1 active site contains a functionally critical cysteine residue (Cys113) with a low predicted pKa, making it highly susceptible to oxidation. We hypothesize that Pin1 is …

Absence Of Phosphoenolpyruvate Carboxylase Atppc3 Increases Sensitivity Of Arabidopsis Thaliana To Cadmium, Ian R. Willick

Electronic Thesis and Dissertation Repository

Phosphoenolpyruvate carboxylase (PEPC) and PEPC kinase (PPCK) catalyze a reaction feeding into the tricarboxylic acid (TCA) cycle, increasing the production of metal-chelating organic acids. Little research has been conducted on PEPC isoenzymes in Cd-stressed plants. Arabidopsis (Arabidopsis thaliana) wild-type and AtPPC1 – AtPPC3 mutants, each lacking one of three PEPC isoenzymes, grown in 0, 1, or 5 µM CdCl₂ were smaller and had increased AtPPC1 – AtPPC3 and AtPPCK1 – AtPPCK2 transcript abundance, relative phosphorylation, and PEPC activity, more so in roots than shoots. Concentrations of oxaloacetate, citrate and total organic acids increased with greater CdCl_{2 …}

Identification Of Cyclophilin Gene Family In Soybean And Characterization Of Gmcyp1, Hemanta Raj Mainali

Electronic Thesis and Dissertation Repository

I identified members of the Cyclophilin (CYP) gene family in soybean (Glycine max) and characterized the GmCYP1, one of the members of soybean CYP. CYPs belong to the immunophilin superfamily with peptidyl-prolyl cis-trans isomerase (PPIase) activity. PPIase catalyzes the interconversion of the cis- and trans-rotamers of the peptidyl-prolyl amide bond of peptides. After extensive data mining, I identified 62 different CYP genes in soybean (GmCYP1 to GmCYP62), of which 8 are multi-domain proteins and 54 are single domain proteins. At least 25% of the GmCYP genes are expressed in soybean. GmCYP1 …

Human Adenovirus E1a Binds And Retasks Cellular Hbre1, Blocking Interferon Signalling And Activating Virus Early Gene Transcription, Gregory J. Fonseca

Electronic Thesis and Dissertation Repository

Upon infection, human adenovirus (HAdV) must block interferon signaling and activate the expression of its early genes to reprogram the cellular environment to support virus replication. During the initial phase of infection, these processes are orchestrated by the first HAdV gene expressed during infection, early region 1A (E1A). E1A binds and appropriates components of the cellular transcriptional machinery to modulate cellular gene transcription and activate viral early genes transcription. We have identified hBre1/RNF20 as a novel target of E1A. hBre1 is an E3 ubiquitin ligase which acts with the Ube2b E2 conjugase and accessory factors RNF40 and WAC1 to monoubiquitinate …

Regulation Of Lipid Homeostasis, Inflammatory Signalling And Atherosclerosis By The Peroxisome Proliferator-Activated Receptor Delta, Lazar A. Bojic

Electronic Thesis and Dissertation Repository

The peroxisome proliferator-activated receptor (PPAR) δ is a ligand-dependent transcription factor that has been implicated in metabolic and inflammatory regulation. The molecular and physiological mechanisms by which PPARδ activation regulates lipid metabolism, inflammatory signaling and protection from atherosclerosis in states of metabolic disturbance such as insulin resistance and dyslipidemia, were investigated in a series of in vitro and in vivo studies. In vitro experiments demonstrated that PPARδ activation inhibits atherogenic lipoprotein-induced lipid accumulation and the associated proinflammatory responses. The primary mechanisms for these effects were increased fatty acid β-oxidation, decreased lipoprotein lipase (LPL) activity, reduced MAPK signaling and improved insulin …

Metabolism And Transcriptional Responses To Asparagine In Arabidopsis Thaliana, Qianyi Zhang

Electronic Thesis and Dissertation Repository

Asparagine aminotransferase transforms asparagine into α-ketosuccinamate, which is further deamidated by an ω-amidase. Serine:glyoxylate aminotransferase, encoded by AGT1 in Arabidopsis, was identified as asparagine aminotransferase. In the roots of 10-day-old Arabidopsis seedlings treated with 20 mM asparagine, AGT1 transcript levels increased by 2-fold while ω-amidase transcripts were decreased by 30%. Recombinant AGT1 had a substrate preference for asparagine when compared with alanine and serine as amino group donors. An ω-amidase candidate gene, AT5G12040, was identified based on amino acid sequence identity with mammalian gene Nitrilase 2. RT-PCR of a T-DNA insertion mutant line showed that ω-amidase expression was abolished …

Dna Hydrolysis And Genome Editing Applications Of Giy-Yig Family Homing Endonucleases, Benjamin P. Kleinstiver

Electronic Thesis and Dissertation Repository

The ability to manipulate complex genomes in a precise manner is essential for studying biological processes in model systems, engineering plant strains for agriculture, or advancing human cellular therapies to treat diseases. Genomic alterations are most efficient when a double-strand DNA break is introduced at the loci where the modification is desired. Different classes of naturally occurring DNA endonucleases, including homing endonucleases, have therefore been explored as candidates for genome modification studies as they target long stretches of DNA. Homing endonucleases are mobile genetic elements whose biological role is to introduce site-specific double-strand breaks into naïve genomes, ultimately resulting in …

Transcriptional Regulation By The Oncogenic Znf217/Corest Complex, Gobi Thillainadesan

Electronic Thesis and Dissertation Repository

The ZNF217 transcription factor is an oncogene found within the 20q13 amplicon and is amplified and overexpressed in many cancers including breast and ovarian. Overexpression of ZNF217 leads to increased cell proliferation, survival, and causes resistance to TGFβ's anti-proliferative effects.

ZNF217 is a core constituent of a transcriptional complex that includes CoREST, HDAC1/2, LSD1, and the CtBP1/2. In this study, I have combined genome-wide biochemical approaches to identify genes directly regulated by ZNF217. I have identified the tumor suppressor and cell cycle inhibitor, p15^ink4b, as a direct target of the ZNF217 complex and demonstrated that ZNF217 represses the …

The Effect Of Glycan Linked To Cationic Peanut Peroxidase On Its Activity, Maha M. Al-Subaie

Electronic Thesis and Dissertation Repository

The roles of heme-, calcium- and glycan moieties on the peanut peroxidase enzyme activity have been previously studied. While the 3-D protein structure is known and some information about the glycan chain is known, due to uneven distribution of the 5 glycans, only a partial understanding of the furthest sugar groups on the 16 glycan chain was available. Now, having used gel-filtration column chromatography followed by lectin affinity column work, I showed that there is a rich array of galactose there. Moreover, I determined that not too many of the sugars are removed in the culture period of 14 days. …

Characterization Of A Tumour Suppressor Function Of Ranbpm, Elnaz Atabakhsh

Electronic Thesis and Dissertation Repository

Ran-binding protein M (RanBPM) is an evolutionarily conserved nucleocytosolic protein that has been proposed to regulate various cellular processes, including protein stability, gene expression, receptor-mediated signalling pathways, cell adhesion, development, and apoptosis. Despite the multitude of functions attributed to RanBPM however, little is known regarding the precise mechanisms by which RanBPM executes these cellular roles. In this work, we seek to address this matter by describing functions for RanBPM in the regulation of apoptotic and pro-survival signalling pathways, and in cellular transformation.

We first identify RanBPM as a pro-apoptotic protein that regulates the activation of the intrinsic apoptotic signalling pathway …

Human Equilibrative Nucleoside Transporter Subtype 1: Structure-Function Analysis Using Cysteine Mutagenesis And Thiol Modifying Techniques, Jamie Park

Electronic Thesis and Dissertation Repository

Human equilibrative nucleoside transporter 1 is the main mediator of bi-directional nucleoside flux and is found ubiquitously. Inhibitor and substrate interactions with ENT1 are known to be affected by cysteine-modifying reagents. Our aim was to investigate the importance of cysteine residues in hENT1 function and identify which residues were sensitive to thiol modification for further application of cysteine scanning mutagenesis on extracellular loop 5. Transporter function was assessed by the binding of [³H]NBMPR and the cellular uptake of [³H]2-chloroadenosine. Treatment of hENT1 with the neutral sulfhydryl-modifier methyl methanethiosulfonate (MMTS) enhanced [³H]NBMPR binding but decreased …

Protein Body Formation In Stable Transgenic Plants Of Nicotiana Tabacum Expressing Elastin-Like Polypeptide And Hydrophobin Fusion Proteins, Sonia P. Gutierrez

Electronic Thesis and Dissertation Repository

Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. However, the use of plants still faces two main limitations: the low accumulation levels of some recombinant proteins and the lack of efficient protein purification methods. Two fusion partners, elastin-like polypeptides (ELP) and hydrophobin I (HFBI) were found to increase the accumulation of recombinant proteins and induce the formation of protein bodies (PBs) when targeted to the ER in transient expression assays. In this study I examined the effect of these tags in stable transgenic plants of two Nicotiana tabacum cultivars when fused to green fluorescent …

Structural Motifs Of Novel Metallothionein Proteins, Duncan E K Sutherland

Electronic Thesis and Dissertation Repository

Metallothioneins (MT) are a family of small cysteine rich proteins, which have been implicated in toxic metal detoxification, protection against oxidative stress, and as a metallochaperone. The most well studied member of the family is the mammalian MT, which consists of two domains: a β-domain with 9 cysteine residues, which sequesters 3 Cd²⁺/Zn²⁺, and an α-domain with 11 cysteine residues, which sequesters 4 Cd²⁺/Zn²⁺. The exact functions of MT are unknown but must relate to its metalation status. Several areas that could lead to the assignment of function include 1) the determination …

Mass Spectrometry-Based Proteomics Analysis Of The Matrix Microenvironment In Pluripotent Stem Cell Culture, Christopher Hughes

Electronic Thesis and Dissertation Repository

The stem cell microenvironment contains soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to effect cellular behavior. Mass spectrometry based proteomics offers the opportunity to directly assay components of extracellular microenvironments, thereby providing a sensitive means for obtaining insight into the stem cell niche. In this study we present the generation and analysis of human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) matrix microenvironments using an MS-based proteomics approach.

One of the primary limitations in the proteomics analysis of hESCs and hiPSCs is the reproducible generation of sufficient cell numbers amenable …

Regulation Of G Protein Signaling By Goloco Motif Containing Proteins, Peishen Zhao

Electronic Thesis and Dissertation Repository

Signal transduction via heterotrimeric G proteins in response to transmembrane G protein-coupled receptors plays a central aspect in how cells integrate extracellular stimuli and produce biological responses. In addition to receptor-mediated activation of heterotrimeric G proteins, during the last few decades, accessory proteins have been found to regulate G protein activity via different mechanisms. Several proteins have been identified that contain multiple G protein regulatory domains. Using various molecular and biochemical approaches, we have characterized the effects of two such proteins, G18 and RGS14, on G protein activity. Both proteins contain a second G protein binding domain in addition to …

Transactivation By Human Adenovirus Early Region 1a-Conserved Region Three, Jailal Ng Ablack

Electronic Thesis and Dissertation Repository

One of the critical functions of human adenovirus (hAd) early region 1A (E1A) protein is to activate transcription of the early viral genes. The largest isoform of E1A contains a unique region termed conserved region 3 (CR3), which includes a Cysteine-4 (C4) zinc finger domain. This region activates viral gene expression by interacting with and recruiting cellular transcription machinery to the regulatory regions of early viral genes. Although this process has been studied at length with hAd type 5 E1A, far less is known about how the E1A proteins from other hAd types activate transcription. There are dramatic differences in …

Identification Of Regions Responsible For The Open Conformation Of S100a10 Using Chimaeric S100a11/S100a10 Proteins, Liliana Santamaria-Kisiel

Electronic Thesis and Dissertation Repository

S100A11 is a dimeric, EF-hand calcium-binding protein. Calcium binding to S100A11 results in a large conformational change that uncovers a broad hydrophobic surface used to interact with phospholipid-binding proteins (annexins A1 and A2), and facilitate membrane vesiculation events. In contrast to other S100 proteins, S100A10 is unable to bind calcium due to deletion and substitution of calcium-ligating residues. Despite this, calcium-free S100A10 assumes an “open” conformation that is very similar to S100A11 in its calcium-bound state (Ca2+-S100A11). To understand how S100A10 is able to adopt an open conformation in the absence of calcium, seven chimeric proteins were constructed where regions …