Browsing by Subject "Department of Biochemistry, Molecular Biology and Biophysics"
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Item Adipocyte Secretion in Metabolic Syndrome(2020) Bell, Marcus; Hertzel, Ann; Bernlohr, DavidMetabolic syndrome is characterized by chronic, low grade inflammation of the adipose organ and increased basal white adipocyte lipolysis. These processes may promote insulin resistance and cell senescence. Dr. Ann Hertzel and Dr. David Bernlohr showed that, in response to lipolysis, adipocytes secrete hundreds of proteins unconventionally (UPS). Among the most abundant of these secreted proteins is fatty acid binding protein 4 (FABP4), a leaderless lipid carrier and known agonist of several mechanisms of insulin resistance, hepatic steatosis, and atherosclerosis. The mechanism of secretion is not well understood, and proteins may be secreted freely or in vesicles. Another notable aspect of lipolytic UPS is its dependence on autophagy, which leads to the hypothesis that it may rely on sphingosine-1-phosphate (S1P) pathway. Additionally, the chronic inflammatory state of the adipose organ may lead to cellular senescence. The molecule 4-hydroxynonenal (4-HNE), a product of lipid peroxidation, may promote cellular senescence via the carbonylation of DNA and proteins. In 2015, Hauck and Bernlohr showed vastly increased intracellular levels of 4-HNE in adipocytes treated with inflammatory cytokines. The passive membrane diffusion of 4-HNE is plausible because it is small and amphipathic yet largely nonpolar. If 4-HNE is secreted from adipocytes, it may act in a paracrine manner and cause local cells to senesce. Adipocyte senescence may result in a decrease in triglyceride storage ability. When this occurs subcutaneously, visceral fat depots become preferred for triglyceride storage – a hallmark progression of obesity and its comorbities. 4-HNE secretion from adipocytes has not been shown in biochemistry literature but is hypothesized to be under lipolytic or inflammatory control due to its high abundance in obese adipocyte cytoplasm. Characterizing the mediating factors of UPS and 4-HNE secretion may show that adipocyte secretions can exacerbate metabolic syndrome and promote cell senescence. Furthermore, showing that adipocytes secrete 4-HNE may introduce an area of study for researchers of aging processes, especially as they relate to obesity and metabolic syndrome.Item Candida albicans Mutagenesis: Response to Stress(2009-04-08) Bruck, David JoachimCandida albicans is a model eukaryotic yeast and an opportunist human pathogen. It generates novel drug resistance through mutation and mitotic recombination. The rate loss of heterozygosity, or rate LoH, is a measure of this mutation and recombination. Previous research has shown that some stresses like the anti-fungal drug fluconazole cause the yeast cells to increase their rate LoH while other stresses do not. On the assumption that fluconazole does not directly affect DNA replication, it is possible that the increased rate LoH is due to a stress response pathway. My UROP project aimed to create a list of computationally curated gene targets using microarray expression data analysis which would then be tested for mutant phenotype rate LoH. The majority of the time allocated to the project was spent doing ortholog searches of gene lists curated from another yeast, Saccharomyces cerevisiae, as well as studying any previous papers where any of the orthologs in question were directly examined.Item Characterizing the Catalytic Activity and Specificity of RNF168 using Targeted Mass Spectrometry and Ubiquitination Stoichiometry-Based Quantitative Analysis(2021) Wei, LaiThe Chen lab developed a new technique that uses quantitative proteomics approach for site-specific ubiquitination stoichiometry analysis, named the Isotopically Balanced Quantification of Ubiquitination (IBAQ-Ub), which is a method that quantifies the absolute abundance of ubiquitination. IBAQ-Ub and in vitro ubiquitination enzymatic assays were conducted to characterize the site-specific ubiquitination dynamics and linkage specificity of RNF168 in DDR(DNA-damage-response).Item Combating HIV: Exploring the Oligomeric Structure of APOBEC3G (A3G)(2010-04-21) Valesano, JohnItem Combating HIV: Exploring the Structure of APOBEC3G (A3G)(2009-04-08) Valesano, JohnThe human APOBEC3G (A3G) protein is a DNA deaminase that can inhibit HIV replication. In most forms of HIV though, the deaminase activity is limited by the viral protein VIF. While recent work has shed light on the three dimensional structure of A3G, little is known about its complete structure and the domains that mediate self-interaction. For instance, inside some types of cells human, A3G forms much larger aggregates with itself, RNA and other proteins, but it is not known how these aggregates are put together. In order to fully understand how A3G interacts with polynucleotides (single-stranded DNA and RNA), how it is inhibited by VIF and how it is regulated within the cell, it is necessary to know how two A3G proteins dimerize. I hypothesized that a distinct site within A3G nucleates or mediates dimerization. To test this hypothesis, a large library of full-length A3G mutants was created. These mutants were then tested in a well-known protein-protein interaction assay called the yeast two-hybrid system. Mutant A3G proteins were pitted against the C-terminal domain of A3G to screen for those that had lost the capacity to dimerize. Candidate dimerization mutants were then sequenced to determine the changes in the nucleotide bases that were responsible. I currently have over ten non-interacting candidates. In the coming months, theses candidates will be tested for their ability to deaminate DNA, dimerize in an independent protein-protein interaction assay and restrict HIV replication in a model cell-based system. My data will help reveal whether the functional unit of A3G is a monomer, a dimer or some more complex polymer.Item Comparative Analysis of Hs6st and Sulf1-mcherry Expression Patterns in Drosophila(2011-04-13) Uk, Samantha; Dang, AnHeparan sulfate proteoglycans (HSPGs) are molecules that are comprised of a core protein modified with heparan sulfate (HS), a negatively charged linear polysaccharide, consisting of uronic acid and N-acetyl-glucosamine (GlcNAc) disaccharide repeats. Generally, these molecules are located on the cell surface and the extracellular matrix. HSPGs have been known to be associated with various biological processes such as growth factor signaling, neuronal development, and cell adhesion. HS chains possess heterogeneous structures, and their diverse patterns of sulfation can determine the binding specificity for certain proteins. 6-O-sulfation of GlcNAc (or GlcNSO3) is the key modification of HS, since it can be dynamically regulated; heparan sulfate 6-sulfotransferase (Hs6st) catalyzes the transfer of sulfate groups of GlcNAc (or GlcNSO3), while heparan sulfate 6-O endosulfatase (Sulf1) removes it. However, how 6-O-sulfation is regulated during animal development remains largely unknown. In this poster, we will present expression analysis of these enzymes in the fruit fly Drosophila melanogaster. We generated a transgenic reporter fly for Sulf1 gene, and its expression pattern was compared with that of Hs6st gene using Hs6st-lacZ enhancer trapline. Our study will provide regulatory mechanisms of HS during the development of Drosophila as well as other multi-cellular organisms.Item Computational Modeling of Protein Kinase A and Comparison with Nuclear Magnetic Resonance Data(2009-10-07) Shi, Lei; Veglia, GianluigiProtein phosphorylation is fundamental in the modulation of myocardial contractility. Sarcoendoplasmic reticulum Ca2+ ATPase(SERCA) removes cytosolic Ca2+ to initiate relaxation, but the regulatory protein, phospholamban(PLN), decreases SERCA’s affinity for free Ca2+. Phosphorylation of PLN by Protein Kinase A (PKA) induces a relief of inhibition on SERCA and augments the rate of SERCA Ca2+ uptake. Here, we studied the interaction between PKA and PLN by nuclear magnetic resonance (NMR), computational docking and molecular dynamics (MD) simulations. Comparative simulations of PKA apo, binary and ternary states were performed, which provided molecular details to understand the mechanism of PKA substrate recognition.Item Determining the Location of Tektins within a Vital Molecular Machine(2011-04-13) Ries, MichaelFlagella and cilia play crucial roles in keeping life moving, one of the most easily recognizable flagella being a spermazoon’s tail. These remarkable molecular machines contain a ring of 9 outer doublet microtubules (hollow tubes, 30 nm in diameter) each composed of protofilaments (PFs). Within each microtubule is a hyper-stable, 3-PF “ribbon” containing the integral, filamentous protein tektin. Two hypotheses exist regarding the location of tektin: (i) the dynein-spoke-tektin filament and (ii) the partition model: (i) suggests that tektin forms one of the three PFs. (ii) suggests that a tektin filament lies on top of the ribbon and is not one of the PFs. In this project ribbons were first isolated from Strongylocentrotus purpuratus (sea urchins). Secondly, they were partially digested by several different enzymes and sized (showing degree of digestion) by gel electrophoresis. Electron microscopy verified that the ribbons were still partially intact. Finally, flanking tubulin fragments still loosely associated with the tektin filament were to be identified by mass spectrometry. By comparing these short fragments of tubulin to their known PF structure, we would be able to deduce whether the tektin filament binds to the sides of the flanking tubulin PFs (as in model i) or binds along the inside surface of the ribbon (as in model ii). The optimal digestion has yet to be achieved; future studies aim to optimize these conditions, through use of different digestive enzymes and digestion times, and subsequently distinguish which model is indeed a more accurate representation of tektin location.Item Engineering a Multi-species Fermentation Platform for Biofuel Production(2011-11) Babson, David; Yeom, Soo-Jin; Schmidt-Dannert, ClaudiaBiodiesel production is an emerging renewable fuel technology. By using microbial consortia, the biosynthetic pathway can be divided-up among multiple microbial populations to create more efficient production processes. The mixed populations are controlled by synthetic interspecies communication circuits, and the consortia metabolism can be engineered to optimize available biomass utilization.Item EPR Analysis of Myosin Structural Dynamics(2009-10-07) Harris, RobertThe structural dynamics of myosin during muscle contraction can be discerned in situ through site directed spin labeling of myosin and electron paramagnetic resonance (EPR) spectroscopy. To achieve in situ measurements, spin labeled myosin regulatory light chain (RLC) is exchanged for endogenous RLC in rabbit psoas fiber bundles. In order to ensure the structural integrity of exchanged muscle fibers, functional measurements must be done before and after exchange. After verifying function after RLC exchange, we can use EPR to measure the orientational dynamics of the RLC in a variety of states during muscle contraction.Item Fluorescence Analysis of the Sarcolipin:SERCA Protein Complex(2009-04-08) Rubin, John E.We have used fluorescence resonance energy transfer (FRET) to identify physical interactions between the sarcoplasmic reticulum Ca-ATPase (SERCA) and one of its regulatory proteins, sarcolipin (SLN), in cardiac and skeletal muscle. The sarcoplasmic reticulum (SR) is an intracellular membrane network found in muscle cells whose function is to uptake, store, and release calcium. SERCA functions to transport calcium into the SR to induce muscle relaxation. Theoretical models predict that SLN monomers regulate SERCA by binding the SERCA transmembrane domain, but SLN monomers also self-associate to form oligomers. To test these models, we expressed fluorescent fusion proteins of SLN and SERCA in Sf21 insect cells using the baculovirus system. Quantitative binding stoichiometries were determined by FRET measurements using live cell microscopy on plates coated with mollusk “glue” protein. FRET results indicate that (1) SLN monomers self-associate to form dimers and (2) SLN monomers interact with SERCA to form a 1:1 heterocomplex. We propose that SLN monomers compete in equilibrium between SLN oligomerization and SERCA binding.Item Gene expression differences between hemangiosarcoma cells in monolayer and non-adherent sphere culture(2011-04-13) Sahli, NathanaelThe cancer stem cell (CSC) theory argues that tumors have a subset of cells that initiate, maintain, and expand cancer in an affected patient. Experimental support for this theory comes from studies that identified sub-populations of cells in a tumor that have the capacity to evade common cancer treatments such as chemotherapy and radiation. Additionally, these same cells exclusively retain the capacity to initiate new disease in xenograft studies. The study of these evasive cells was initially challenging as they differentiate in standard serum-containing culture medium where they grow as a monolayer. In the past decade, methods for culturing stem cells using a serum-free medium has allowed CSCs to be maintained, where they form non-adherent multicellular spheres. Here, we cultured canine hemangiosarcoma (HSA) in both a multicellular sphere and standard monolayer system to compare gene expression using real time qRT-PCR. In our system, the monolayer cultures are a useful surrogate for differentiated tumor cells (the bulk of the tumor), while the serum-free sphere-derived cells are a surrogate for in vivo CSC. Here, we investigate differences in gene expression between these two cultures systems. The genes chosen for study have been shown to be up-regulated in CSCs from various other cancers, or normal stem cells, with minimal expression in differentiated cells. We found gene expression differences between cultures conditions which will allow it to be utilized in the study of hemangiosarcoma as well as possibly other cancers with a CSC.Item Identification of the fla4 Mutation in Chlamydomonas reinhardtii(2009-04-08) Tank, DamienThe unicellular green alga Chlamydomonas reinhardtii is commonly used as a model organism to study flagella and their associated proteins. The fla4 mutant exhibits normal flagellar function at a permissive temperature of 21C, but sheds its flagella at a restrictive temperature of 32C. Transformation with large insert BAC clones had previously identified a candidate FLA4 gene. I confirmed the identity of the FLA4 gene by transformation with a smaller subclone to rescue the mutant phenotype. The flagella of two transformed strains functioned normally at the restrictive temperature. Using reverse transcriptase-polymerase chain reactions (RT-PCR), gel electrophoresis, and sequence comparisons between wild-type and fla4 DNA, I determined the complementary DNA (cDNA) sequence of the wild-type FLA4 gene and identified the specific mutation in fla4. The predicted amino acid sequence corresponds to a conserved TPR repeat protein. This protein shows significant similarity to proteins found in many higher organisms, such as human and mouse. In animals, this protein has been associated with defects in nervous system development, but its cellular function is unknown. Further study of its function in C. reinhardtii may provide insight into its role in higher organisms.Item Injury by human complement causes large membrane lesions that reseal in IL-4-treated porcine endothelial cells(2011-04-13) Yeh, AlexCurrently, successful xenotransplantation is restricted to theoretical conception due to the fact that an organism’s innate immune response rejects any tissue or organs transplanted from a different species. The complement system is a key component of this response in rejecting foreign substances, and mitigating the effects of this system could potentially revolutionize medical transplants. Complement system activation in an organism creates lesions in the membranes of foreign cells, leading to lysis and cell death. IL-4 is a cytokine, or a cell-signaling molecule, that seems to have a protective property against complement. Porcine endothelial cells first incubated in IL-4 exhibit decreased cell death after treatment with human complement, but the exact mechanism of protection is still unknown. A combination of the Neutral Red assay and the LDH assay were used to study cell recovery after complement treatment. Fluorescent microscopy was also used, in which labeled dextrans of different sizes were incubated with the cells along with complement. Should the mechanism of IL-4 protection be correctly identified, it may have potential uses in mitigating the innate immune response and the rejection of organs in xenotransplantation.Item Investigating Brachypodium distachyon as a Model System for Plant Biofuels Research(2011-04-13) Lavell, AnastasiyaPlant biomass has been proposed for the production of biofuels. Grasses are an attractive source of biomass due to their fast growth rate and abundance. The mechanical and chemical breakdown of grass cell walls releases simple sugars that are stored in the cellulose and hemicellulose of the plant. These can then be fermented to produce a variety of fuels. Brachypodium distachyon (Brachypodium) possesses many desirable qualities of a model system such as small genome, diploid accessions, small stature, and short life cycle. Brachypodium is a powerful tool for investigating how to make grasses better biofuel crops. Although Brachypodium is already being used for such research, a great deal of information still needs to be gathered on this grass to fully exploit it as a model for biofuel research. Two distinct features of Brachypodium were investigated here. First, the ability of different Brachypodium genotypes to regenerate back to healthy fertile plants through the tissue culture method was studied. A second study examined the effect of cold treatment (vernalization) on plant height, biomass, days to spike emergence, and the amount of fermentable sugars released from mature stems. The comparison of regeneration efficiencies of five Brachypodium genotypes showed that each genotype was able to form compact embryogenic calli (CECs) and regenerate green shoots from callus tissue at different efficiencies. CEC formation efficiencies ranged from 22% to 86%. Efficiencies for regeneration of healthy fertile plants will be gathered in the future as plants mature. The ability of these Brachypodium genotypes to form CECs is significant because CECs are necessary for transforming plants to study how grasses can be altered for more efficient biofuel production. Vernalization of three Brachypodium genotypes for different time periods (0, 7, 14, 21, and 28days) showed that with increased vernalization time, height and days to spike emergence were reduced. The most dramatic reduction in days to spike emergence occurred between zero and two weeks. Data on fermentable sugar release of different Brachypodium genotypes will be provided in the future by a collaborating laboratory.Item Investigating the Induction and Purification of OGG1 Protein and Its Role in DNA-Protein Cross-Linking(2010-04-21) Nguyen-Tran, Thuy DuongDNA-protein cross-links (DPC’s) occur when a protein reacts with a DNA strand. DPC’s can lead to cell death if it is not repaired due to its potential to interfere with cellular processes such as DNA replication and transcription because it causes a bulky distortion in the DNA double helix. Various chemical agents, such as 1,2,3,4-Diepoxybutane (DEB), are known to induce DPC’s because of their alkylating functions. When uncontrollably created in cells, DPC’s have a harmful effect, but they can have a therapeutic function if they are specifically induced in cells and controlled. Thus, with its potential lethalness to cells, DPC’s have been studied as a possible cancer treatment by trying to target its production in tumor cells and observing its biological activity. To better understand, DPC’s, this project focused on the creation of DPC’s using 8-oxoguanine glycosylase (OGG1) and the OG oligonucleotide duplex (OG). Specific focus was placed on the induction and purification of the OGG1 protein and cross-linking it to OG.Item Mechanism of Disease-Causing Missense Mutations in Dystrophin(2009-04-08) Lee, AnnDuchenne muscular dystrophy (DMD) is a fatal, x-linked disease that affects 1 in every 3,500 live born males. DMD is caused by the loss of the protein dystrophin due to genetic mutation. Dystrophin is abundant at the cell membrane of muscle cells, where its function is to stabilize the plasma membrane against contraction-induced membrane damage by binding to cytoskeletal f-actin filaments and the transmembrane protein dystroglycan. A small percentage of DMD cases are caused by missense mutations where the change in a single amino acid can cause severe disease. This disease can be caused by dystrophin not being able to bind its intracellular partners or by misfolding of the dystrophin protein, which can lead to degradation or insoluble aggregates. I investigated this aggregation as a possible mechanism for the pathogenesis of DMD missense mutations. I specifically worked with five missense mutations: K18N, L54R, L172H, Y231N, and T279A. These particular mutations are located in the actin-binding domain on the N-terminus of dystrophin. Of the fifteen known missense mutations in DMD patients, nine of them are found in this region. The missense mutations in this area can cause disease in one of two ways. The mutations can cause the protein to fold improperly, which leads to the aggregation and the loss of its localization to the membrane. The mutations can also render the protein unable to bind actin properly, despite normal folding and transportation of the protein to the membrane. For this project, I compared the levels of insoluble protein for various types of mutant dystrophin to the levels of wild type (WT) dystrophin (data provided by D.M. Henderson) to determine whether the mutations cause misfolding. I hypothesized that disease-causing missense mutations in the N-terminal actin binding domain of dystrophin cause misfolding and increase the level of insoluble dystrophin in vivo.Item Molecular Interactions of Sarcolipin and Phospholamban Using Fluorescence Resonance Energy Transfer (FRET) in Live Cells(2010-04-21) Rubin, John E.We have used fluorescence resonance energy transfer (FRET) microscopy to measure the binding affinities of four protein interactions in muscle to elucidate the binding events that occur during the formation of the SERCA super-inhibitory complex. Calcium is transported into the sarcoplasmic reticulum (SR) during muscle relaxation by the sarcoplasmic reticulum Ca- ATPase (SERCA), which is separately regulated by two transmembrane proteins, sarcolipin (SLN) and phospholamban (PLB). It has been proposed that when SERCA, SLN, and PLB are all expressed in the same muscle cell, the three proteins bind together in a super-inhibitory ternary complex, which decreases SERCA calcium transport by 50%. A key intermediate to this proposed ternary complex is the SLN:PLB heterocomplex. In my project, FRET microscopy was used to confirm the presence of the SLN:PLB heterocomplex and to directly quantitate the degree physical interaction between the two proteins in live cells. For comparison, FRET microscopy was also used to quantify SLN:SLN, SLN:SERCA, and PLB:SERCA interactions. Average FRET was directly calculated for each protein:protein interaction on a cell-to-cell basis. In addition, a Michaelis-Menten binding model and non-linear Hill fitting were used to calculate the dissociation constant for each protein interaction and the intrinsic distance between fluorescent probes. FRET results indicated that SLN and PLB form a low affinity heterodimer in cells with a distance of 4.6 nm between subunits. FRET results also show that SLN:SLN has the highest binding affinity of the four interactions while SERCA:SLN and SERCA:PLB have medium binding affinities relative to SLN:PLB and SLN:SLN. We propose that SLN and PLB first bind independently to SERCA and then bind to each other to induce the super-inhibitory SERCA ternary complex.Item Optimization of Engineered Cardiovascular Tissue(2009-04-08) Meier, Lee A.The ultimate goal of the field of tissue engineering is the ability to develop engineered tissues that exhibit physiologically relevant dimensions and mechanical properties such that utilization in a clinical setting is possible. While the most common approaches in this field have resulted in significant progress toward this goal's realization, in order to fully understand the cell developmental processes that guide tissue formation in vitro, as well as in vivo, a modified approach is necessary. The field of systems biology is emerging with the hope of solving this problem. Its purpose is to better understand complex cellular signaling interactions through mathematical modeling in order to develop statistical correlations within large sets of data obtained through interrogation of the biological system of interest. Developing statistical correlations between externally induced cellular signaling events and the resulting tissue phenotype can aid in elucidation of a predictive method for understanding how a cell population will respond to varying degrees of stimulation. The question posed is: if one knows the cell/tissue culture input stimulation(s), can the resulting tissue qualities be accurately predicted? A statistical method known as discriminant partial least squares regression is commonly employed for such analyses. This type of analysis relies on construction of a matrix (X) describing the signaling events induced within a population of cells in response to varying degrees of stimulation, as well as a second matrix (Y) describing the observed cellular response in terms of expressed phenotype. It is hypothesized that a solution to the expression Y=f(X) provides a well-defined description of the connected signaling events within the system of interest. Least squares regression methods have been empirically proven effective for such previously mentioned purposes. Neonatal human dermal fibroblasts (NHDF) and Fischer rat vascular smooth muscle cells (SMC) are of primary interest for this experiment. Two-dimensional cell monolayers were utilized initially and the experimental procedure will be later extrapolated to vascular, three-dimensional, fibrin-based tissue equivalents, in particular: tunica media-equivalents (ME). The Flexcell International FX-4000 cell culturing system was used to subject cultured monolayers to periods of cyclic distention/strain, as well as varying degrees of growth factor and supplement stimulation. Following distention and biochemical stimulation, cultures were lysed and the soluble fraction isolated. Subsequently, total protein content was determined and the samples were enriched for their phosphoproteins. Mass spectrometry was then employed to quantify the enzymatic activity within individual populations of cells. This analytical medium has elucidated significant phosphoprotein mass profile distinction between differentially stimulated cell populations. Statistical correlations between the input stimulation events and the resulting cell population's qualities such as the phosphoprotein mass profiles, total collagen and elastin content, as well as total cell number will be determined. From this, a cell-culture stimulation paradigm of steepest-ascent towards developmentally- and mechanically-optimized tissue-equivalents will be employed.Item The PKC Inhibitor Gö 6976 Blocks C-Type Natriuretic Peptide Activation of Guanylyl Cyclase B(2009-10-07) Lou, XiaoyingThis study characterizes the effects of the widely used protein kinase C inhibitor, Gö 6976, on NPR-B guanylyl cyclase activity as a means to identify its inhibitory mechanisms.