Browsing by Subject "Biochemistry, Molecular Bio, and Biophysics"
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Item Actin Isoforms in neuronal structure and function(2011-07) Cheever, Thomas R.The actin cytoskeleton plays critical roles in nearly every aspect of neuronal development and function. During these processes, the localized polymerization of actin is one mechanism employed to carryout crucial tasks for normal neuronal function. While the activity of actin binding proteins is generally thought to be the primary mediator of spatially restricted actin polymerization, another prominent mechanism involves the local translation of β-actin, one of two actin isoforms expressed in neurons. The localized translation of β-actin has been shown previously to be essential for growth cone guidance in cultured neurons. Additionally, defects in the localization of β-actin have been implicated in the motor neuron disease Spinal Muscular Atrophy (SMA). However, no study to date has directly examined the role of β-actin in a mammalian in vivo system. Although the functions of β-actin were thought to be critical for all neurons, the work described in this thesis indicates that specific functions of β-actin are surprisingly confined to select populations in the central nervous system (CNS). β-actin is not required for motor axon regeneration or motor neuron function, but is required for the proper structure of the hippocampus, cerebellum, and corpus callosum, as well as hippocampal-associated behaviors. Thus, the work described here provides the first direct demonstration of specific roles for β-actin in vivo and presents a model to translate provocative findings in cell culture to the mammalian CNS.Item Adipocyte protein carbonylation and oxidative stress in obesity-linked mitochondrial dysfunction and insulin resistance.(2011-09) Curtis, Jessica MarieCarbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. The antioxidant enzyme glutathione S-transferase A4 (GSTA4) catalyzes a major detoxification pathway for such reactive lipids but its expression was selectively down regulated in the obese, insulin resistant adipocyte resulting in increased protein carbonylation. The effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. Mitochondrial functions in adipocytes of lean or obese GSTA4 null mice were significantly compromised compared to wild type controls and were accompanied by an increase in superoxide anion. Silencing GSTA4 mRNA in cultured adipocytes resulted in increased protein carbonylation, increased mitochondrial ROS, dysfunctional state 3 respiration and altered glucose transport and lipolysis. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4- silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1 alpha subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production and a reduction in membrane potential without changes in mitochondrial number, area or density. These results suggest protein carbonylation plays a major instigating role in mitochondrial dysfunction and may be a linked to the development of insulin resistance in the adipocyte.Item APOBEC3 proteins restrict mobile and foreign DNA(2009-08) Stenglein, Mark DanielA cell's DNA provides its operating instructions. Consequently, cells rigorously maintain their DNA. At the same time, cells must prevent foreign DNA from persisting, or risk having their operations hijacked. This thesis describes how APOBEC3 proteins help fulfill this double imperative, by limiting the replication of mobile genetic elements, and by triggering the destabilization of foreign intracellular DNA. The APOBEC3s are a family of DNA modifying enzymes that convert deoxycytidines to deoxyuridines. Introducing uridines into DNA can alter the sequence of genes or regulatory elements, in essence removing information content from the molecule. Such an alteration also often leads to the destabilization and degradation of the DNA. Previously, APOBEC3s had been shown to act on a relatively limited subset of viral and retrotransposon DNA replication intermediates. The major contribution of this work is to expand the range of biologically relevant APOBEC3 substrates to include LINE-1 retrotransposons and foreign intracellular DNA in general. LINE-1s are the only retrotransposons currently active in humans, and their mobilization can cause disease by a variety of mechanisms, the most straightforward of which is by inserting in or near a gene and disrupting its function. Expression of several APOBEC3 proteins reduces the rate of LINE-1 retrotransposition in human cells. Less retrotransposed LINE-1 DNA accumulates in these cells, suggesting that the block occurs prior to retrotransposon integration. The APOBEC3s therefore limit the mutagenic potential of LINE-1 elements. Foreign intracellular DNA is inherently dangerous and is often associated with microbial infection. In recognition of this danger, sensors within cells detect foreign DNA, but, previously, little was known about mechanisms that respond to DNA detection to mediate its clearance. This thesis demonstrates that APOBEC3 family members are induced following DNA detection and destabilize foreign DNA. This defense system is likely to have evolved to respond to intracellular microbial DNA, but it may also diminish the efficacy of processes such as genetic engineering and gene therapy. Finally, this thesis explores the molecular basis of the differential sub-cellular localization of the APOBEC3 proteins. Sequestering APOBEC3 proteins in different cellular compartments may be a way to regulate the DNA substrates to which they have access. This thesis therefore offers several insights into a family of proteins that modify potentially harmful DNA in order to protect the well-being and proper function of cells.Item Beyond the allosteric role of Fructose-2,6-bisphosphate: exploring its effects on gene expression and signal transduction.(2009-09) Khan, Salmaan AhmedIn diabetes, insulin is either not available or not effective in suppressing glucose output by the liver. Enhancing hepatic glucose flux through glycolysis by raising fructose-2,6-bisphosphate (F26BP) levels ameliorates the diabetic phenotype in type 1 and type 2 diabetic mouse models (WU et al. 2001b; WU et al. 2002). This is attributed to the well characterized allosteric effects F26BP has on glucose metabolism enzymes. Recent studies have determined that raising F26BP levels has an additional role in regulating gene expression and signal transduction proteins (WU et al. 2005; WU et al. 2004). In terms of glucose metabolism, raising F26BP levels stimulates glucokinase gene expression and inhibits glucose-6-phosphatase gene expression, thus, also favoring enhanced glycolysis. These effects are not limited to glucose metabolism, as raising F26BP levels in type 2 diabetic mice leads to decreased weight gain and adiposity as well as a decrease in lipogenic enzyme gene expression. To fully characterize this new role of raising F26BP levels on gene expression, a microarray together with iTRAQ proteomic analysis was carried out. Through functional analysis of the microarray biological pathways were identified that changed when F26BP levels were raised in type 2 diabetic mice. In this way a wider array of lipid and cholesterol metabolism genes was shown to be down-regulated. Additionally, several other novel effects of F26BP on gene expression were elucidated. Comparison of the microarray and proteomic datasets elucidated that 87% of the protein expression changes were concomitant with gene expression changes. It is our hypothesis that some of these gene and protein expression changes are mediated through F26BP effect on the insulin signaling pathway component, Akt. Raising F26BP levels in a streptozotocin-treated mouse model (type 1 diabetic) leads to increased Akt levels and phosphorylation at serine-473 (WU et al. 2004). Based on this, experiments were carried out to determine the mechanism by which F26BP activates Akt and interacts with the insulin signaling pathway. Raising F26BP in cultured cells enhances insulin's effect on Akt and this effect requires phosphatidylinositol-3 kinase to be active. However, different from what is observed in vivo, in cell culture F26BP alone is not able to affect Akt phosphorylation.Item A biochemical and biophysical study of dystrophin.(2011-07) Henderson, Davin MichaelThe primary role of a muscle cell is to contract and produce force that moves an organism. A vast majority of a muscle is made of the proteins in contraction machinery and nearly all energy utilized by the cell is consumed in this process. However, an equally important but substantially smaller portion of the muscle cell is dedicated to the preservation of cell membrane integrity. The costamere is an elaborate matrix of cytoskeleton associated and transmembrane proteins that form a support lattice between the plasma membrane and contractile apparatus. The dystrophin glycoprotein complex (DGC) is a structurally important member of the costamere and has been shown to link microtubules, thin and intermediate filaments of the cytoskeleton with major components of the extracellular matrix. In the DGC, dystrophin is responsible for attachments with intracellular cytoskeletal components and the transmembrane protein dystroglycan. One of the most common diseases afflicting muscle is Duchenne muscular dystrophy (DMD), which is caused by mutations in the gene encoding the protein dystrophin. The focus of my thesis is to better understand the biochemical and biophysical properties of dystrophin. Specifically, I investigated the actin binding properties of dystrophin in the context of its functional domains as well as the consequences of disease causing missense mutations localized to actin-binding domain 1 (ABD1). Additionally, I characterized the biophysical properties of internally truncated dystrophin proteins under development for treatment of DMD. It has been twenty years since dystrophin was hypothesized to bind actin and even today we are learning more about this fascinating interaction. My thesis expands our understanding of the dystrophin-actin interaction in three ways. First, I showed that full-length dystrophin interacts with the actin isoforms expressed in muscle with equivalent affinities. Second, I showed that the thermally stable C-terminal domain of dystrophin is required for full actin binding activity. Third, in collaboration with the Thomas lab, we showed that dystrophin and utrophin uniquely alter the physical properties of actin filaments. Disease causing missense mutations in the dystrophin gene are scattered in many functional domains but we chose to study a cluster localized to ABD1 with hope that we would find amino acids important for actin binding. We hypothesized that mutations in ABD1 would disrupt actin binding and therefore lead to disease though loss of an essential interacting partner. However, no mutation dramatically disrupted actin binding but instead lead to loss of thermal stability and protein aggregation. My thesis work was the first to show evidence that protein stability and aggregation may play a role in the pathogenesis of dystrophinopathies. DMD currently has no effective treatment but many promising therapies are being pursued. Many laboratories are pursuing therapies for DMD and multiple techniques are being pursued including adeno-associated viral gene therapy, protein replacement therapy, exon skipping therapy and stop codon read though therapy. For gene therapy and protein therapy, the size of the dystrophin or utrophin coding sequence has been reduced by deletion of internal domains, which retains important N- and C-terminal ligand binding sites. I set out to test the stability of internally deleted therapy proteins to ensure that no unwanted structural perturbations were caused by internal deletion. Additionally, I tested a set of N-and C-terminal truncations of dystrophin and a dystrophin related protein, utrophin for comparison to internally deleted versions of these proteins used in therapy. I found that the thermal stability of utrophin was uniform from N- to C-terminus and that internal deletion did not affect protein stability. I also found that the N-terminal half of dystrophin had a lower thermal stability compared to the C-terminal half and, to our surprise, internally deleted dystrophin proteins showed marked thermal instability and aggregation.Item Biochemical and functional characterization of fatty acid transport proteins.(2009-07) Wiczer, Brian MichaelThe adipocyte fatty acid transport proteins (FATPs), FATP1 and FATP4, have been implicated in both lipid influx and storage and understanding their role in adipose tissue would gain insight into the persistence of metabolic disorders, such as type 2 diabetes. FATP1 was previously determined to be an acyl-CoA synthetase and work described in this thesis additionally explored the acyl-CoA synthetase activity of purified FATP4. FATP4 was found to be a more robust acyl-CoA synthetase than FATP1. Through the use of RNAi in cultured adipocytes, silencing the expression of either FATP1 or FATP4 results in cellular phenotype demonstrating improved insulin responsiveness. Interestingly, silencing FATP1 abolished insulin-stimulated long-chain fatty acid (LCFA) influx, whereas silencing FATP4 had no effect on LCFA influx despite its higher activity. Furthermore, the expression of FATP1 was demonstrated to be important for the activation of the AMP-activated protein kinase during insulinstimulated LCFA influx. In addition to the cytoplasmic localization of FATP1, it was also found to exhibit mitochondrial localization. Further analysis demonstrated a novel role in the regulation of TCA cycle function and mitochondrial energy metabolism, in part, through the interaction of FATP1 with the 2-oxoglutarate dehydrogenase complex, a rate-limiting step in the TCA cycle. This work shines light on how FATPs may play broader roles in metabolism that previously appreciated and the potential implications associated with such roles.Item Biophysical characterization of membrane proteins and antimicrobial peptides by solution and solid-state NMR spectroscopy.(2011-03) Verardi, RaffaelloMembrane proteins and antimicrobial peptides represent two diverse and challenging classes of macromolecules to characterize at the molecular level. They are linked by the interaction with the lipid bilayer of the cell membrane. Within the lipid bilayer, membrane proteins are involved in vital biochemical processes such as ion transport, signal transduction and cell adhesion. Antimicrobial peptides are a broad class of polypeptides produced by all living organisms, representing the first line of defense against bacterial infections. They work by selectively targeting the bacterial membranes and subsequently killing the cell by a variety of mechanisms such as membrane disruption, membrane potential dissipation and enzyme inactivation. Although very important, membrane proteins and antimicrobial peptides are underrepresented in terms of available high-resolution structural information compared to water-soluble proteins and this limits the current understanding of how they work in living cells. In this thesis I summarize my contribution towards the elucidation of the high-resolution structures of the integral membrane protein phospholamban and the mechanism of action of two important antimicrobial peptides (LL37 and distinctin) by a hybrid solution and solid-state nuclear magnetic resonance spectroscopy approach. These results provide new insights and methodologies to study and understand how key membrane proteins and antimicrobial peptides elicit their function.Item Cardiac calcium transport regulation probed by electron paramagnetic resonance spectroscopy.(2010-07) Torgersen, Kurt DanielMuscle contraction and relaxation is regulated by calcium flux between the sarcoplasmic reticulum and the cytoplasm. Subsequent to muscle contraction, calcium must be sequestered to the sarcoplasmic reticulum in order for muscle relaxation to occur. The sarco-endoplasmic reticulum Ca-ATPase (SERCA) is a P-type ATPase embedded in the SR membrane which uses ATP hydrolysis to pump calcium back into the SR lumen to facilitate muscle relaxation. In cardiac muscle, SERCA activity is regulated by phospholamban (PLB) a 52-residue integral membrane protein which exists in a dynamic equilibrium between monomeric and pentameric species. Previous data have shown that monomeric PLB is the primary regulator of SERCA activity but recent publications have proposed that the PLB pentamer may also bind to and inhibit SERCA activity. This inhibition can be relieved by phosphorylation of PLB at Ser16, although the mechanism is not known. Electron Paramagnetic Resonance (EPR) experiments were designed to test two proposed models of the PLB pentamer, the pinwheel and bellflower. Dynamics data using the TOAC amino acid spin label showed that, like the monomer, the pentamer is in a dynamic equilibrium between ordered (T) and dynamically disordered (R) states, with the T state being predominant. Accessibility of spin labels attached to the cytoplasmic domain to the lipid bilayer showed that, like the monomer, the pentamer cytoplasmic domains strongly interact with the lipid bilayer surface. Finally, pulsed EPR (DEER) experiments measuring long range distances between spin labels attached to the cytoplasmic domain showed a bimodal distance distribution with centers at 3 and 5 nm. All of these data support the pinwheel model. To investigate SERCA binding and phosphorylation affects on PLB dynamics, a monomeric mutant, AFA-PLB, was spin labeled with TOAC either the 11 position in the cytoplasmic domain or 36 in the transmembrane domain. Conventional EPR measurements showed that phosphorylation induced and order-to-disorder conformational change in the cytoplasmic domain and that SERCA preferentially binds the PLB R state. Phosphorylation of SERCA bound PLB resulted in a disorder-to-order conformational change, suggesting that pPLB is still bound to SERCA. Conventional dynamics from 36-TOAC in the transmembrane indicated a stable helix which was unaffected by phosphorylation or SERCA binding. Dipolar EPR measurements revealed that phosphorylation of PLB in the absence of SERCA induces oligomerization and that SERCA destabilizes the pPLB oligomer. Saturation Transfer EPR data which measures the rotational diffusion of PLB in the lipid bilayer supported the conclusion that phosphorylation of PLB in the absence of SERCA induces oligomerization and showed directly that phosphorylated PLB is still bound to active SERCA. These data support the model that phosphorylation dependent relief of SERCA inhibition does not require dissociation of the SERCA-PLB complex, but is rather the result of a structural change in the complex.Item ChREBP: insights into the mechanism of action by glucose.(2010-06) Davies, Michael NealCarbohydrate Response Element Binding Protein (ChREBP) is a glucose-responsive transcription factor that activates genes involved in de novo lipogenesis in mammals. The current model for glucose activation of ChREBP proposes that increased glucose metabolism triggers a cytoplasmic to nuclear translocation of ChREBP that is critical for activation. However, we find that ChREBP actively shuttles between the cytoplasm and nucleus in both low and high glucose in the glucose-sensitive β cell-derived line, 832/13. Glucose stimulates a three-fold increase in the rate of ChREBP nuclear entry, but trapping ChREBP in the nucleus by mutagenesis or with a nuclear export inhibitor does not lead to constitutive activation. In fact, mutational studies targeting the nuclear export signal of ChREBP also identified a distinct function essential for glucose-dependent transcriptional activation. From this, we conclude that an additional event independent of nuclear translocation is required for activation. Glucose regulation of ChREBP has been mapped to its conserved N-terminal region of 300 amino acids, designated the MondoA Conserved Region (MCR). Within the MCR, five domains (MCRs 1-5) have a particularly high level of conservation and are likely to be important for glucose regulation. We carried out a large-scale deletion and substitution mutational analysis of the MCR domain of ChREBP. This analysis revealed that MCRs 1-4 function in a concerted fashion to repress ChREBP activity in basal (non-stimulatory) conditions. Deletion of the entire MCR 1-4 segment or combining four specific point mutations (Quad mutant) throughout this region lead to a highly active, glucose-independent form of ChREBP. However, deletion of any individual MCR domain and the majority of point mutations throughout MCRs 1-4 rendered ChREBP inactive. These observations suggest that MCRs 1-4 interact with a factor required for activation and that this interaction occurs after repression is relieved. This possibility is supported by the observation that the MCR 1-4 region can compete for activity with wild type ChREBP and the derepressed Quad mutant in both basal and stimulatory conditions. Thus, the MCR domains act in a complex and coordinated manner to regulate ChREBP activity in response to glucose.Item Defining AMD disease mechanisms: a comparative analysis of proteins and mitochondrial DNA(2010-08) Karunadharma, Pabalu Pussellage RanminiAge-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. Current treatments are limited due to our inadequate understanding of the pathogenic events leading to AMD. Early clinical symptoms occur in the retinal pigment epithelium (RPE), suggesting RPE as the potential site of defect in AMD. This research evaluated the RPE proteome and mitochondrial DNA (mtDNA) to test the hypothesis that molecular changes in the RPE contributes to AMD. Human donor eyes categorized into four progressive stages of AMD were utilized in these investigations. Two proteomic analyses using 2D gel electrophoresis and mass spectrometry were performed to define changes in the RPE proteome. In the first proteomic study, analysis of the mitochondrial proteome revealed significant changes that suggested potential damage to mtDNA with AMD. These results prompted an analysis of mtDNA lesions associated with aging and AMD. These results suggest a potential link between mt dysfunction due to increased mtDNA damage and altered proteins and AMD pathology. In the second study, we tested the hypothesis that mt dysfunction is communicated to the nucleus via retrograde signaling and consequently alters the protein profile to reflect a major shift in metabolism and stress response. Our results suggest not only adjusted metabolism, response to stress and cellular redox regulation but also show major differences in the protein profile with AMD compared to aging. In summary, our investigations distinguished between normal and pathologic aging by identifying key macromolecules and pathways affected with each process. Furthermore, our data indicate a potential link between mitochondrial dysfunction and AMD pathology, thus providing a point of intervention for the treatment of AMD.Item Distinct developmental functions for cytoplasmic actin isoforms.(2010-12) Bunnell, Tina M.Actins are among the most highly expressed proteins in eukaryotes and play a critical role in most cellular processes. In mammals there exists six different actin isoforms, of which only the cytoplasmic βcyto- and γcyto-actins are ubiquitously expressed. Remarkably, the cytoplasmic actins differ at only 4 out of 375 amino acids and have been exactly conserved from birds to mammals. It has been postulated that βcyto- and γcytoactin have distinct biological functions; therefore, to test this hypothesis we generated null alleles of the Actb and Actg1 genes. Characterization of the resulting isoformspecific null animals demonstrates that βcyto-actin but not γcyto-actin is essential for embryonic viability. While γcyto-actin is largely dispensable for embryonic development, it does confer growth and survival advantages, as evidenced by the fact that γcyto-actin null embryos exhibited mild developmental delays and decreased postnatal survival Furthermore, γcyto-actin null primary mouse embryonic fibroblasts (MEFs) had a mild growth deficiency and a slight increase in apoptosis, despite total actin levels being maintained. In contrast to γcyto-actin null mice, βcyto-actin null mice were early embryonic lethal, indicating that βcyto-actin is an essential gene required for embryogenesis. The lethality in βcyto-actin null mice is likely due to defects in cell growth and migration as these processes were severely impaired in βcyto-actin knockout primary MEFs. Together, the distinct phenotypes observed in βcyto- and γcyto-actin knockout mice and cells demonstrate that while βcyto- and γcyto-actin can compensate for each other to a limited extent, they also have unique biological functions.Item Effect of sequence context on the formation and repair of DNA adducts.(2009-08) Guza, Rebecca ClareTobacco smoke contains over sixty known carcinogens and following exposure can lead to the formation of DNA adducts. Persistence of nucleobase adducts in DNA can lead to polymerase errors, mutations in critical gene, and tumor initiation. However, a specialized repair protein, O6-alkylguanine DNA alkyltransferase (AGT), recognizes and repairs the O6-alkyl-dG adducts. Major targets for mutations in smoking induced lung cancer are the K-ras proto-oncogene and the p53 tumor suppressor gene. Mutational hotspots in the p53 gene are concentrated at endogenously methylated CG dinucleotides in exons 5-8. These sites contain 5-methylcytosine (MeC), which is known to alter DNA structure and stability. We hypothesized that MeC may contribute to carcinogenesis by influencing the formation and repair of tobacco carcinogen-DNA adducts within critical genes. In our studies, mass spectrometry based methodologies were used to analyze the kinetics of AGT repair of O6-Me-dG and to investigate the mechanisms of MeC mediated effects on DNA adduct formation at MeC:G base pairs. We found that the rate of AGT-mediated repair of O6-Me-dG lesions is influenced by local sequence context and in particular, can be mediated by endogenous cytosine methylation. MeC can also mediate the formation of N2-BPDE-dG adducts by influencing the formation of pre-covalent and intercalative complexes between DNA and BPDE. This work contributes to our understanding of the role of sequence context and endogenous cytosine methylation in the formation and repair of DNA adducts induced by tobacco carcinogens.Item Enzyme catalyzed perhydrolysis, molecular basis and application(2011-10) Yin, Delu (Tyler)Enzyme catalyzed perhydrolysis converts a carboxylic acid or ester to a peracid. In the former reaction, the amount of peracid generated is thermodynamically controlled (Keq = 3) – while in the latter, the reaction is kinetically controlled, thus a higher concentration of peracid can be generated. Enzymes that catalyze perhydrolysis of carboxylic acids share high sequence similarity and are thought to use an esterase-like mechanism. Alternatively, carboxylic acids can also use a non-covalent mechanism, such as those used by hydroxynitrile lyases. To test whether carboxylic acid perhydrolases use an esterase-like mechanism, we identify a key covalent intermediate by mass spectrometry that can be attributed to an esterase-like mechanism but not a non-covalent mechanism. We also find that carboxylic acid perhydrolases are good catalysts for hydrolysis of peracetic acid, suggesting that their natural role is to degrade peracids generated as by-products in a living organism. Next, we determine how perhydrolases increase the rate of perhydrolysis. Carboxylic acid perhydrolases increase the rate of perhydrolysis by either increasing the selectivity for hydrogen peroxide or lower the activation barrier towards acylenzyme formation. We measure the selectivity of hydrogen peroxide using wild-type Pseudomonas fluorescens esterase (PFE) and L29P PFE (a model carboxylic acid perhydrolase). The L29P PFE variant is less selective for hydrogen peroxide than the wild-type despite having higher perhydrolysis activity. We measure the rate of acyl-enzyme formation using isotope exchange of acetic acid in H218O/H216O. The L29P PFE variant catalyzes the isotope exchange rate faster than the wild-type. Thus, carboxylic acid perhydrolases favor the formation of acyl-enzyme from carboxylic acids. We find that carboxylic acid perhydrolase (L29P PFE) does not catalyze ester perhydrolysis for accumulating high concentrations of peracetic acid. Instead, wild-type PFE and a new variant, F162L PFE accumulate up to 130 mM of peracetic acid. We measure kinetic parameters and show that hydrolysis of peracetic acid limits maximum accumulation. The F162L PFE variant minimizes hydrolysis of peracetic acid by lower ing the Km and increasing the kcat for ethyl acetate hydrolysis. The kinetic parameters are also used to predict the maximum amount of peracetic acid that can be accumulated. The F162L PFE variant is used to improve the efficiency of lignocellulose pretreatment from a previously published result using wild-type PFE. Enzymatically generated peracetic acid reacts converts lignin into smaller and more soluble lignin pieces. The chemoenzymatic process is further improved by forming peracetic acid in a biphasic layer which allows the reuse of enzyme. The pretreatment reaction conditions were also optimized by increasing the temperature to 60 °C and reducing the reaction time to 6 hours.Item From functional metagenomics to unique synthetic expression strategies in iron-reducing bacteria.(2012-05) Gonzalez, Tanhia DenysCellulose and hemicellulose are renewable sources of fermentable sugars. The use of fermentable sugars for the production of alternative energy sources (i.e. ethanol, butanol, etc.) is an attractive solution to alleviate the shortage and high prices of petroleum. Cellulases and hemicellulases are the two groups of glycosyl hydrolases responsible for breaking down the polysaccharide component of biomass into their respective sugar moieties. The enzymatic hydrolysis of cellulose and hemicellulose has relied on enzymes originally produced by culturable organisms. This thesis describes the use of metagenomics coupled to high-throughput screening techniques to identify glycosyl hydrolases originally encoded by uncultured organisms. The findings of this thesis include the identification and biochemical characterization of a unique endoglucanase. Besides catalyzing the hydrolysis of soluble and insoluble cellulosic substrates, this endoglucanase exhibited a domain architecture that has not been previously reported in the literature. This thesis also describes two different strategies to engineer the surface of (Fe+3)-reducing bacteria. These expression systems are a valuable tool for studying the cellular respiration of Geobacter and Shewanella. Furthermore, they have practical applications in the area of whole-cell biocatalysis in microbial fuel cells. The first strategy involved using an autodisplay system to engineer the cell envelope of Geobacter and Shewanella. The autodisplay system translocated a functional β-galactosidase enzyme to the cell envelope of G. sulfurreducens and S. oneidensis. Furthermore, this system proved to be an effective tool for catalyzing reactions in electrochemical cells using biofilms of G. sulfurreducens cells. The second strategy exploited the use of in-frame fusions with the c-type cytochrome OmcZ to translocate a recombinant protein to the outer membrane and extracellular matrix of Geobacter sulfurreducens. This is the first time that the c-type cytochrome OmcZ has been used to engineer biofilms of Geobacter sulfurreducens.Item From structure and dynamics to novel therapeutic development for muscular dystrophy.(2012-07) Lin, Ava YunDystrophin is defective in Duchenne (DMD) and Becker (BMD) muscular dystrophies, which are debilitating X-linked diseases that currently have no cure. Dystrophin links the actin cytoskeleton at its N-terminus and a glycoprotein complex (DGC) embedded in the sarcolemma at its C-terminus, apparently providing mechanical stability to the muscle during contraction. Due to the large size (427 kD) and filamentous nature of dystrophin, studies of its function and attempts to develop effective therapeutics have developed slowly, despite intensive efforts. Utrophin (395 kD) is a homolog of dystrophin that has shown therapeutic promise in mdx mice, which lack dystrophin. Utrophin is endogenously expressed in the cytoskeleton of fetal and developing muscle but is replaced by dystrophin as the muscle matures (8-10). Both dystrophin and utrophin belong to the spectrin superfamily of actin-binding proteins, which carry out diverse functions in the cytoskeleton of most cells. Of the many proteins included in this superfamily, dystrophin and utrophin are among the least studied in terms of structural dynamics, limiting the understanding of their function at the sarcolemma. In order to target the root of dystrophin malfunction in muscular dystrophy, we need to better understand the native functions of dystrophin and utrophin. Lack of structural information about dystrophin and its interactions adds to the complexity of tying clinical presentations to the diverse disease-causing mutations, and hinders therapeutic advancement in gene or drug therapy. There are numerous mouse-model studies, but there are varied results across several parameters tested, and no construct or drug has been found that restores normal muscle force in the mdx mouse. Exon-skipping morpholinos are expensive to produce with variable delivery and efficacy to muscle groups and require a customized oligo design for each mutation, making it difficult to test them individually in mouse models. In order to (a) understand disease mechanisms and (b) design better therapies rationally, we need more fundamental information about the structures and interactions of specific regions of dystrophin and utrophin. That is the goal of this project.Item Identification of the transcription factor ZEB1 as a novel modulator of adiposity.(2009-09) Saykally, Jessica NicoleObesity and its subsequent metabolic disorders have become global health problems. While this is largely due to environmental influences, the propensity to gain weight also has a significant genetic component. In an effort to identify genes that contribute to obesity, several genome-wide scans on obese patient populations have been performed. One consistent location that displayed linkage to obesity is chromosome 10p11-12. A likely candidate gene within this region is the TCF8 gene, which encodes the ZEB1 transcription factor. To test whether TCF8 is an anti-obesity gene, DNA from obese patients was genotyped using single nucleotide polymorphisms throughout the TCF8 genetic locus. Logarithm of the odds for linkage of TCF8 to childhood obesity was high in two regions. Sequencing of a subset of the patient DNAs revealed a polymorphism that results in an amino acid change within the coding region of 50% of the patients. More tests will be required to determine whether the polymorphism has a functional consequence. In addition, TCF8+/- and wild type (WT) C57BL/6 mice were fed a high fat diet or regular chow diet for 20 weeks and their body weights, body composition, and metabolic parameters measured. TCF8 +/- female mice were significantly heavier on both diets due to an increase in fat. Increased adipose mass was the result of increased adipocyte size and was sufficient to cause metabolic consequences. Interestingly, this phenotype was not observed in male or female mice treated with the specific anti-estrogen Faslodex, suggesting that ZEB1 is mediating some of estrogen’s anti-obesity effects. Expression of several known estrogen-regulated genes important in lipolysis and lipogenesis measured in TCF8 +/- and WT suggests that ZEB1 modulates the flux of lipids in adipocytes. This thesis identifies TCF8 as an anti-obesity gene in mice and potentially in humans. Loss of one copy of the TCF8 gene is sufficient to increase adiposity and subsequent metabolic consequences. This is a novel observation as no one has previously proposed a role for ZEB1 in adipose tissue. In addition, this data contributes to our understanding of sexual dimorphism in metabolism.Item Identifying novel roles for the immunoproteasome in the retina.(2010-10) Hussong, Stacy AnnImmunoproteasome is a proteasome sub-type that is known to produce antigenic peptides for MHC class I presentation. However, immunoproteasome is present in the immune-privileged brain and retina and is upregulated with disease in human retina and injury in mouse retina and brain, suggesting functions unrelated to its role in the immune system. The goal of this thesis is to define novel roles for the immunoproteasome in the retina. Potential functions of the immunoproteasome were defined by comparing the stress response of wild-type and knock-out mice missing one (lmp7-/-(L7)) or two (lmp7-/- /mecl-1-/-(L7M1)) of the three immunoproteasome subunits. Aging was used as a model system for chronic stress. Chronic peroxide exposure in cultured retinal pigment epithelial (RPE) cells developed from wild-type mice was used as an additional stress model. In wild-type retinas and RPE cells, upregulation of immunoproteasome was observed in response to both models of chronic stress. To determine the consequence of eliminating immunoproteasome, the retinas and RPE cells from KO mice were examined.L7M1 retina had significantly elevated levels of photoreceptor apoptosis that further increased with age. In addition, L7M1 cell lines were more susceptible to oxidantinduced death. Together these data suggest immunoproteasome is protective against oxidative stress. The localization of immunoproteasome to the outer plexiform layer in wild-type retina suggested a role in retinal function. Electroretinography was used to test the hypothesis that immunoproteasome is required for maintaining normal visual transmission. Data indicated that immunoproteasome-deficient mice had a decreased bipolar cell response as compared to wild-type. Evaluation of several retinal synapse proteins by Western blot revealed no significant difference in protein content across strains. In addition, gross retinal morphology and bipolar cell density were not different. In conclusion, immunoproteasome-deficiency causes a decrease in visual transmission but the mechanism is still unclear. In summary, these data provide compelling evidence that immunoproteasome has a role in retinal stress response, specifically in protecting against oxidative stress. Furthermore, immunoproteasome-deficient mice have a decreased bipolar cell response as measured by ERG. Altogether, data from this thesis strongly support the hypothesis that immunoproteasome has additional functions in the retina that do not involve immune function.Item Improving the detection of carbonylated peptides by mass spectrometry via solid-phase hydrazide enrichment and selective labeling with Oxygen-18 (18O)(2010-01) Roe, Mikel RobertProtein carbonylation is a post-translational oxidative protein modification known to alter protein function and impair cellular mechanisms. It is a relatively complex modification, characterized by a variety of structurally distinct reactive carbonyls that target a number of amino acid residues and originate via several different oxidative mechanisms. While identification of specific carbonylated proteins by mass spectrometry has provided insight regarding the protein pathways and complexes affected, the specific sites of carbonyl modification, necessary for determining the oxidative mechanisms involved as well as for explaining any associated functional consequences, are not routinely identified due to the relatively low abundance of carbonylated proteins. To address this issue, a number of methods for enriching carbonylated peptides have been developed, all of which involve derivatization with bulky reagents that often complicate the identification of peptides by tandem mass spectrometry. As an alternative to these label-based approaches, I have developed a label-free method for enriching carbonylated peptides that is based on their selective capture and controlled release from a novel solid-phase hydrazide reagent (SPH). The value of the SPH reagent method is demonstrated using a yeast lysate treated with the reactive lipid carbonyl 4-hydroxynonenal (HNE), where the use of pulsed-Q-dissociation (PQD) and neutral-loss triggered MS/MS/MS was employed for the first time to assist the identification of HNE-modified peptides by mass spectrometry. To further improve the confidence by which carbonylated peptides are identified via mass spectrometry, a novel 18O-labeling method that selectively introduces an 18O molecule into the carbonyl oxygen of carbonylated peptides was developed. The resulting 18O isotope signature enables carbonylated peptide precursor ions and carbonylated MS/MS fragment ions to be tracked in the full-scan MS and MS/MS spectra, respectively, thus providing an independent validation of the MS/MS spectra matched to carbonylated peptides by proteomic database searching. The value of 18O-labeling for both improving the accuracy and measuring the efficiency of database-based identification of carbonylated peptides is demonstrated in an HNE-treated lysate from rat skeletal muscle homogenate. In conclusion, the development of the SPH reagent and the 18O-labeling method are useful tools for identifying carbonylated peptides in complex biological mixtures and represent important steps forward in the field of redox proteomics.Item Informing the Oral Squamous Cell Carcinoma Biomarker Search by Exudate Proteomics(2013-04) Kooren, Joel AllanOral cancer is the sixth most common cancer worldwide ahead of Hodgkin's lymphoma, leukemia, brain, stomach, or ovarian cancers, with about 41,000 Americans being diagnosed annually. More than 90% of oral cancers are oral Squamous cell carcinomas (OSCC). While the overall 5-year survival rate is about 60%, the survival rate when diagnosed early is higher than 80%. Currently the standard for diagnosis of OSCC is early visual detection of a suspicious oral lesion followed by scalpel biopsy with histology. However, the invasiveness, expense, and required expertise involved prevents consistent application on at risk individuals. Chapter 1 discusses the methods that are being investigated for sampling and discovering biomarkers of OSCC that address some of these limitations. Protein biomarkers contained in samples collected non-invasively and directly from at-risk oral premalignant lesions (OPML) would address current needs in a uniquely targeted fashion. Chapter 2 of this thesis describes work evaluating the potential of a novel method using commercial PerioPaper absorbent strips for the collection of oral lesion exudate fluid coupled with mass spectrometry based proteomics for OSCC biomarker discovery. This research focuses on demonstrating the feasibility of using oral lesion exudates in proteomic research, exploring the proteome of exudate samples, discriminating between exudates collected from clinically different sources, with supplemental table 1 showing which proteins distinguish healthy and OPML sources. Furthermore, to ensure that the best possible marker candidates are selected given clinical sample availability, multiple methods were explored enable and improve quantitative proteomic analysis of exudates in chapter 3 (Identified proteins in supplemental files 2 and 3). Our label-free quantitative proteomics strategy analyzed paired control and OPML exudates (figure 8), identifying differentially abundant proteins between sample types. Next, we selected several [exudate] differentially abundant proteins for testing in while saliva, comparing their relative abundance levels in healthy, OPML and oral Squamous cell carcinoma (OSCC) subjects. Two proteins, CK10 and A1AT, showed differences in saliva. Our results provide a demonstration of the value of tissue exudate analysis for guiding salivary biomarker discovery in oral cancer, as well as providing promising biomarker candidates for future evaluation.Item Insight into the oxygen activation mechanism by Rieske dioxygenases through kinetic, spectroscopic and mutagenesis studies.(2010-04) Chakrabarty, SarmisthaRieske dioxygenases catalyze the first step in the degradation of aromatic hydrocarbons. They facilitate dioxygen bond cleavage with insertion of both O atoms as hydroxyl groups into the aromatic substrate; this produces a non-aromatic cis-diol. Here studies of the chemical and regulatory mechanisms of benzoate 1,2-dioxygenase (BZDO) and naphthalene 1,2-dioxygenase (NDO) are described. These multicomponent enzymes consist of an (αβ)3 oxygenase component in addition to a reductase and, in the case of NDO, ferredoxin components that mediate the electron transfer from NAD(P)H. The oxygenase component contains a Rieske [2Fe-2S] cluster and a non-heme mononuclear Fe center, which is the site for the O2 activation and product formation. Transient kinetic and spectroscopic studies of BZDO show that electron transfer from the Rieske cluster to an adjacent Fe center across the subunit boundary occurs in 3 phases due to the presence of at least 2 and probably 3 different types of active sites. These differences in nominally identical active sites are proposed to originate from structural changes related to redox state-mediated regulation. This is demonstrated by a Magnetic Circular Dichroism study with NDO that reveals changes in iron coordination number and geometry controlled by the redox state of the Rieske cluster and the presence of substrate. Mutagenesis studies of the essential subunit interface residue Asp205 in NDO show that it is unlikely to be the sole mediator of electron transfer and regulatory conformational change as proposed by others. The nature of the reactive oxygen intermediate formed at the Fe site was probed using the radical clock substrate probes norcarane and bicyclohexane. They show that monooxygenase chemistry by NDO occurs via a substrate radical, implicating formation of a novel HO-Fe5+=O reactive state that may also pertain to dioxygenase chemistry.