Browsing by Subject "Biochemistry, molecular bio, and biophysics"

Now showing 1 - 7 of 7
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    Accumulation of unligated Okazaki fragments induces PCNA ubiquitination at lysine 107 and Rad59-dependent replication fork progression
    (2012-07) Nguyen, Dang Hai
    DNA ligase I, encoded by the CDC9 gene in Saccharomyces cerevisiae, is an essential enzyme that catalyzes the ligation of newly synthesized DNA on the lagging strand called Okazaki fragments. In humans, approximately 30 million Okazaki fragments are synthesized during every S phase and require further processing prior to DNA ligation. An individual harboring DNA ligase I mutations exhibited growth retardation, sunlight sensitivity, severe immunosuppression and developed lymphoma, indicating a link between defects in Okazaki fragment maturation and cancer predisposition. How cells monitor and suppress such accumulation of DNA damage that arises due to defective Okazaki fragment processing is unclear. Using S. cerevisiae as a model system, we uncovered a novel and conserved ubiquitination pathway that targets proliferating cell nuclear antigen (PCNA) at lysine 107 when DNA ligase I activity is inhibited. The modification at K107 is catalyzed by the E2 variant Mms2 together with Ubc4 and the E3 ubiquitin ligase Rad5. Most importantly, this signal is crucial to activate the S phase checkpoint, which promotes cell cycle arrest. In support of this notion, a pol30K107 mutation alleviated cell cycle arrest in cdc9 mutants. To determine whether PCNA ubiquitination occurred in response to nicks or the lack of PCNA-DNA ligase interaction, we complemented cdc9 cells either with wild-type DNA ligase I or Chlorella virus ligase, the latter of which fails to interact with PCNA. Both enzymes reversed PCNA ubiquitination, arguing that the modification is likely triggered directly by nicks. To further understand how cells cope with nicks during replication, we utilized cdc9-1 in a genome-wide synthetic lethality screen and identified RAD59 as a strong negative interactor. cdc9 rad59 double mutants did not alter PCNA ubiquitination but enhanced phosphorylation of the mediator of the replication checkpoint, Mrc1, indicative of increased replication fork stalling. Thus, Rad59 promotes fork progression when Okazaki fragment processing is compromised and counteracts PCNA-K107 mediated cell cycle arrest.
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    Characterization of the conformational states of phospholamban and their roles in regulation of SR Calcium-ATPase
    (2012-12) Gustavsson, Bengt Martin
    Membrane proteins constitute 30% of the human genome but represent only a small fraction of the known three-dimensional protein structures. In this thesis I describe the characterization of the membrane protein complex between sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLN). SERCA drives cardiac muscle relaxation by active transport of Ca2+ ions into the SR. PLN is a small membrane protein that consists of a helical trans-membrane domain connected to a cytoplasmic domain through a short loop, and inhibits SERCA through intra-membrane interactions. The cytoplasmic domain of PLN is in equilibrium between a helical, membrane-associated T state and an unfolded, membrane-dissociated R state. Here, I summarize the work to probe the structures of the T and R states and elucidate the role of the conformational equilibrium in regulation of SERCA. Using solution and solid state NMR in combination with biochemical assays I show that the structures of T and R state but not their relative populations are conserved in different lipid environments and sample conditions. Furthermore, the T/ R equilibrium has a central role in SERCA regulation and is crucial to relieve the inhibition of the enzyme. These findings provide new insights into SERCA/PLN function and offer a unique view of the role of conformational equilibria and multiple conformational states in membrane protein structure and function.
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    Conformational changes in actinin-type actin binding domains: probing actin-induced structural dynamics in dystrophin and utrophin using EPR spectroscopy.
    (2014-12) Crain, Jonathan
    The underlying cause of Duchenne and Becker muscular dystrophies is a lack of functional dystrophin, a large multidomain protein. Dystrophin is normally expressed in muscle, where it links the extracellular matrix to the cortical actin cytoskeleton via a complex of associated proteins. Dystrophin, and its autosomal homologue utrophin, connect with the actin cytoskeleton through two F-actin binding domains, including an N-terminal "actinin-type" actin binding domain (ABD).In addition to dystrophin and utrophin, actinin-type ABDs are found in a large number of proteins. Nonetheless, the actin binding mechanism remains poorly understood: x-ray crystallography and electron microscopy have produced conflicting models. Electron paramagnetic resonance (EPR) spectroscopy, especially double electron-electron resonance (DEER), can be used to distinguish between these models or to build new models. In this thesis, I present data from DEER experiments which suggest that actinin-type ABDs of dystrophin and utrophin adopt unexpected conformations in solution.
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    Developing new enzymatic catalysts by resurrecting ancestral alpha/beta hydrolases
    (2014-09) Mooney, Joanna Lynn
    In our daily lives, we use items created by synthetic chemistry. In some cases, traditional chemical synthesis of these items requires harsh solvents, extreme temperatures, and results in large amounts of waste generated by side reactions. Developing enzymatic catalysts is a possible solution to this problem because enzymes are more selective, easy to dispose of, and react at ambient conditions. Modern day specialist enzymes are thought to have evolved from ancient generalists that catalyzed several reactions promiscuously. Our lab has reconstructed a number of ancestral enzymes from extant members of the alpha/beta hydrolase family. We have screened these enzymes for promiscuous reaction and substrate activities. The aldol and nitroaldol reactions are very important for the catalysis of many pharmaceuticals and commodity chemicals. One of our reconstructed ancestral enzymes catalyzes a nitroaldol reaction at a higher rate than modern day enzymes. We have also identified what is potentially a partial aldol reaction.
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    In vitro evolution of artificial enzymes: method development and applications
    (2014-09) Haugner III, John Christian
    Artificial enzymes have the potential to aid in the production of pharmaceuticals and facilitate basic biomedical research. There are two methods for making artificial enzymes: rational design and de novo selection. Rational design utilizes detailed knowledge of enzyme catalysis to design an enzyme active site, and then introduces this active site into a protein. However, due to the limited understanding of protein folding and structure-function relationships this approach is still extremely challenging and far from routine. In contrast, we utilize a directed evolution approach to isolate de novo artificial enzymes from a large library of protein variants by in vitro selection. Each of the trillions of proteins in a library are tested in a single experiment to determine if any have the desired activity. The artificial enzymes are created when the library is made so a high quality library is important for success. My thesis research focuses on two goals: (1) Construct a library built on the robust (alpha/beta)8 barrel enzyme scaffold for future enzyme selections and (2) Characterize a thermostable artificial RNA ligase and develop an application for this enzyme. The (alpha/beta)8 fold is used to catalyze a wide range of chemical reactions in nature. We used this fold to create a library containing > 1014 unique proteins by replacing loops of the catalytic face with randomized codons via PCR. Small sub-libraries were subjected to a protease-based folding selection to improve library quality by enriching for folded sequences. The final folding-enriched library contained > 1012 folded proteins representing an up to 50-fold improvement relative to a control library. These libraries will provide a valuable source of new enzymes for future in vitro selections. The previously generated artificial RNA ligases join 5'-triphosphate RNA to the 3'-hydroxyl of a second RNA substrate; a reaction not observed in nature. However the enzymes were also highly dynamic, which prevented the solving of the protein structure by NMR or X-ray crystallography. A more structured enzyme, called ligase 10C, was isolated by performing the ligase selection at 65°C and its structure was solved revealing a novel primordial fold. Here, we describe the detailed biochemical characterization of ligase 10C. Using a variety of RNA substrates, we also determined how ligation rates change with sequence composition revealing an enzyme with broad sequence specificity. We developed a method for the specific ligation and sequencing of 5'-triphosphorylated RNA. These results highlight ligase 10C as an attractive tool for the selective isolation of 5'-triphosphate RNA from a complex mixture, something which is difficult with current methods.
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    Plant phenylpropanoid biosynthesis in Escherichia coli: engineering novel pathways and tools
    (2014-10) Bloch, Sarah E.
    Plant phenylpropanoid natural products are important in the discovery of safe and effective therapeutics. Most plant natural products cannot be economically mass produced via extraction from plant tissue or chemical synthesis. In recent decades, engineering microbes to carry out the biosynthesis of plant natural products has emerged as a powerful technology. The goal of this thesis was to expand the capabilities of microbial biosynthesis of plant phenylpropanoids in Escherichia coli through exploring novel biosynthetic pathways and metabolic engineering tools. I first explored the biosynthesis of valuable lignans in E. coli, establishing random oxidative radical coupling through overexpression of a laccase and attempting to show stereoselective coupling by a dirigent protein. I also designed and built a biosynthetic pathway for rosmarinic acid, a valuable hydroxycinnamic acid ester, showed pathway bottlenecks and limitations, and identified future optimization strategies. I have also begun a project to better understand cargo protein encapsulation within bacterial microcompartments and to develop their utility as a means of spatially organizing metabolic pathways. This work has contributed significantly to the field of microbial metabolic engineering and has laid the groundwork for future economically viable production platforms.
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    Studies on the regulatory mechanism of the ULK1 complex in the induction of autophagy
    (2012-10) Cao, Jing
    Autophagy, an evolutionarily-conserved cellular process through which organelles and macromolecules are degraded in the lysosome, is induced under nutrient starvation or other unfavorable growth conditions. Unc51-like kinase 1 (ULK1) is a serine/threonine protein kinase that plays a key role in the autophagy induction process, but how ULK1 is regulated by cellular signals for induction of autophagy and how ULK1 regulates the downstream processes in autophagy remain poorly understood. ULK1 interacts with Atg13, focal adhesion kinase family interacting protein of 200 kD (FIP200) and Atg101 to form a large protein complex involved in early steps of the autophagy induction process. To better understand the function of the ULK1 complex, my thesis work has sought to identify binding proteins of the complex. Through a yeast two hybrid screen using a human fetal brain cDNA library with Atg13 as bait, a protein named MCF.2 cell line derived transforming sequence-like 2 (MCF2L2) was identified. Through co-immunoprecipitation and in vitro binding assay, MCF2L2 was determined to directly interact with Atg13 via its N-terminal region independently of ULK1. Knockdown of MCF2L2 inhibited the formation of autophagosome and autophagy flux and led to accumulation of p62/sequestosome-1, a protein degraded through autophagy. Knockdown of MCF2L2 also suppressed the aggregation of WD-repeat protein interacting with phosphoinositides-1, an autophagic isolation membrane marker. MCF2L2 contains a putative Rho-guanine nucleotide exchange factor (GEF) domain in the middle and has a sequence similarity to MCF2L and MCF2, the well-known Rho-GEFs. MCF2L2 overexpression induced a moderate increase in the active forms of Rho GTPases and MCF2L2 colocalized with actin related protein 3, the actin nucleation factor that is regulated by Rho GTPases, implying that MCF2L2 potentially contains GEF activity. MCF2L2 knockdown partially suppressed the distribution of Atg9 from trans-golgi network to the cytoplasm in response to starvation, a process that may depend on actin cytoskeleton. Combined, these results suggest that MCF2L2, as a component of the ULK1 complex, might play an important role in mediating signal transduction between the actin cytoskeleton and autophagy induction.