Molecular Biology and Biochemistry - Theses, Dissertations, and other Required Graduate Degree Essays

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Caspase regulation of autophagy in Drosophila melanogaster

Date created: 
2013-04-08
Abstract: 

Autophagy is an evolutionary conserved process whereby intracellular components are sequestered and delivered to lysosomes for degradation. Autophagy acts as a cell survival mechanism in response to stress, such as starvation, and also engages in a complex relationship with apoptosis. Understanding the crosstalk between autophagy and apoptosis is important, as it plays a critical role in the balance between survival and death, and has important implications in both normal development and human diseases. To better understand the crosstalk between autophagy and apoptosis, I examined the role of the Drosophila melanogaster effector caspase Dcp-1 in starvation-induced autophagy during mid-oogenesis. I confirmed that Dcp-1 positively regulates starvation-induced autophagic flux in degenerating mid-stage egg chambers, and does so in a catalytically dependent manner. Dcp-1 candidate interactors/substrates, identified previously, were analyzed using in vitro autophagy assays to elucidate potential mechanisms related to Dcp-1-mediated autophagy. I identified 13 novel Dcp-1-associated regulators of starvation-induced autophagy, including the chloride intracellular channel protein Clic, the heat shock protein Hsp83, and the mitochondrial protein SesB. In vivo analyses revealed that Clic and Hsp83 act as negative regulators of autophagic flux following starvation during Drosophila oogenesis. Further investigation into the possible mitochondrial-related role of Dcp-1 in autophagy revealed that Dcp-1 partially localizes within the mitochondria where it functions to regulate mitochondrial network morphology and ATP levels, demonstrated both in vitro and in vivo during mid-oogenesis. Moreover, I found that the pro-form of Dcp-1 interacts with the adenine nucleotide translocase SesB, and as such, Dcp-1 does not cleave SesB but rather affects its stability. In addition, I identified SesB as a novel negative regulator of autophagic flux during mid-oogenesis. Depletion of ATP or reduction of SesB levels rescued the autophagic defect in Dcp-1 loss-of-function flies, and genetic interaction studies revealed that SesB acts downstream of Dcp-1 in the regulation of autophagy. In conclusion, I found that non-apoptotic caspase activity is an important molecular mechanism underlying autophagy regulation and mitochondrial physiology in vivo, and have provided a foundation for further analyses involving Dcp-1-associated regulators of starvation-induced autophagy.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Sharon Gorski
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Discovery of a thiamin-utilizing α-keto acid decarboxylase ribozyme: Implications for RNA’s role in primordial metabolism

Author: 
Date created: 
2013-09-19
Abstract: 

Vitamins are hypothesized to be relics of an RNA World, and likely participants in an RNA-mediated primordial metabolism. If catalytic RNAs could harness vitamin cofactors to aid their function, in a manner similar to enzymes, it would enable ribozymes to catalyze a much larger set of chemical reactions. The cofactor thiamin diphosphate, a derivative of vitamin B1 (thiamin), is used by enzymes to catalyze difficult metabolic reactions, including decarboxylation of stable α-keto acids such as pyruvate. Here I report a ribozyme that uses free thiamin to decarboxylate a pyruvate-based suicide substrate (LnkPB). Thiamin conjugated to biotin was used to isolate catalytic individuals from a pool of random sequence RNAs attached to LnkPB. Analysis of a stable guanosine adduct obtained via digestion of an RNA sequence (clone dc4) showed the expected decarboxylation product. Discovery of a prototypic thiamin-utilizing ribozyme has implications for RNA's role in orchestrating early metabolic cycles.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Dipankar Sen
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Probing inhibitory contacts between the regulatory and catalytic domains of CTP:phosphocholine cytidylyltransferase (CCT) using Transition metal ion Fluorescence Resonance Energy Transfer (tmFRET)

Author: 
Date created: 
2014-05-30
Abstract: 

CCT catalyzes the rate-limiting step in phosphatidylcholine synthesis. CCT is activated when its lipid-induced amphipathic helix (domain M) binds to PC-deficient membranes and silenced when the auto-inhibitory motif (AI) within domain M binds helix αE in the catalytic domain. tmFRET was used to probe inter-domain interactions. Monobromobimane (donor) was conjugated to a cysteine engineered in αE or AI. My objective was to determine if a native di-histidine motif (89HSGH92) in the active site could function as a Cu2+ (acceptor) binding site. Fluorescence quenching occurred at Cu2+ concentrations above 10-5 M but persisted when the binding site was compromised via histidine protonation, H89S mutation, or competition with CDP-choline. Cu2+-quenching also persisted when domains M and C where dissociated by membrane binding. These results suggest that 89HSGH92 is not an effective Cu2+ binding site and quenching was likely collisional. Future work will require engineering a di-histidine motif elsewhere in the catalytic domain.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Rosemary B. Cornell
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Using Isothermal Titration Calorimetry to Characterize the Ca2+ Binding Properties of Cardiac Troponin C and Slow Skeletal-specific Troponin C in Zebrafish

Author: 
Date created: 
2014-04-16
Abstract: 

Slow skeletal-specific TnC (ssTnC, tnnc1b), found only in teleosts, and cardiac troponin C (cTnC, tnnc1a) are both expressed in zebrafish (ZF) heart in a temperature- and chamber-specific pattern. The focus of this study is to determine the Ca2+ binding affinities (KCa) of the regulatory site in ZF cTnC and ssTnC, and the thermal stability of each protein. Isothermal titration calorimetry was used to determine the KCa of the N- terminal domain of ZF cTnC and ssTnC at 8°C, 18°C and 28°C. The results for both TnCs show an increase in KCa with increasing temperature. The melting temperature (Tm) of each protein was also determined from thermal melting curves. The results show that ZF cTnC is slightly more stable with a Tm of 2°C higher. The data obtained provide functional insight into the Ca2+ binding properties of ZF TnC. It also provides a better understanding of teleost TnC sub-functionalization for chamber-specific expression seen in ZF.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Glen Tibbits
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Development of functional tools to study the role of O-GlcNAc in tau biology

Date created: 
2013-01-21
Abstract: 

The O-GlcNAc post-translational modification involves the attachment of single N-acetyl-D-glucosamine residues to serine and threonine of nucleocytoplasmic proteins of multicellular eukaryotes. The microtubule associated-protein tau has been suggested to be extensively O-GlcNAc modified and to compete with tau phosphorylation. Tau becomes hyperphosphorylated in Alzheimer’s disease (AD) which leads to the formation of the characteristic aggregates seen in the brains of AD patients. The reciprocal relationship between tau O-GlcNAcylation and tau phosphorylation suggests that if O-GlcNAc levels rise then phosphorylation levels should decrease. If O-GlcNAc levels can be increased in vivo, hyperphosphorylation of tau might be, therefore, antagonized by the increased levels of O-GlcNAc. One way to increase O-GlcNAcylation of tau is to inhibit the enzyme which removes O-GlcNAc, a glycoside hydrolase referred to as OGA. Existing OGA inhibitors were unsuitable for use in vivo. For this reason, a potent, selective, and stable inhibitor, termed Thiamet-G, was developed in the laboratory. Treatment with Thiamet-G increased O-GlcNAc levels in both cells as well as in the mouse brain. These substantial increases in O-GlcNAc, resulted in decreased levels of tau phosphorylation at sites of phosphorylation that are implicated in AD. Collaboratively, I also found that Thiamet-G treatment results in prevention of neurodegeneration in the JNPL3 mouse model and further observed that this effect does not appear to arise by prevention of hyperphosphorylation but more likely by blocking the aggregation of tau. This proposal is further supported by experiments showing that O-GlcNAc modification of tau impairs the aggregation of truncated and full length human tau in vitro. In order to study the site-specific effects of tau O-GlcNAcylation several sites of tau O-GlcNAc were mapped collaboratively and the major site of tau O-GlcNAc was found to be Ser400. I developed a site-specific Ser400 O-GlcNAc tau antibody and find that this site of glycosylation plays a key role in the aggregation of tau in vitro. The tools described herein should prove useful in further clarifying the role of O-GlcNAc in tau biology and also may serve to clarify OGA as a target with therapeutic potential.

Document type: 
Thesis
File(s): 
Senior supervisor: 
David Vocadlo
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis/Dissertation) Ph.D.

Functional aspects of ciliary maintenance in Caenorhabditis elegans

Author: 
Date created: 
2013-09-20
Abstract: 

Primary cilia are cellular antennae found on many cell types in metazoans. Their biogenesis and maintenance is critical throughout lifespan of an animal to support signal transduction pathways essential for development, and physiological processes such as vision and olfaction. Intraflagellar transport (IFT) is a process that is required to form and maintain cilia. Studies in Chlamydomonas reinhardtii and Caenorhabditis elegans have revealed several components required for ciliogenesis and IFT, but the function and mechanism of many of these proteins are poorly understood. In this dissertation, I identify and characterize two genes, che-10 and dyf-18, that maintain ciliary function at least in part by modulating IFT. I identified CHE-10 as the rootletin ortholog in C. elegans. Rootletin is an evolutionarily conserved protein that exists as polymerized striated rootlets, a cytoskeleton-like structure associated to the cilium-nucleating basal bodies, or as non-filamentous form associated to the ciliary base. Similar to its disruption in mouse model, che-10 mutants initiate ciliogenesis but the cilia degenerate over time. I showed that rootletin maintains cilia by modulating the assembly, motility and flux of IFT components. I also demonstrated that rootletin is essential for the stability of the axoneme, the transition zone, which forms a ciliary gate, and the basal bodies. Finally, I present evidence that the molecular basis of these defects may be due to inefficient delivery of ciliary components and organization at the periciliary membrane compartment, leading to cilium degeneration. DYF-18, a C. elegans CCRK-related Ser-Thr kinase, was uncovered in a screen for genes expressed during ciliogenesis. I show that DYF-18 is expressed in all ciliated sensory neurons. Similar to C. reinhardtii, disruption of DYF-18 leads to ciliary length defects. Finally, I demonstrated that dyf-18 mutants have abnormal accumulation of key IFT components. Specifically, OSM-5 is at the base of cilia and OSM-3 kinesin accumulates between and middle and distal segments of the axoneme. Intriguingly, in spite of the loss of OSM-3 kinesin in the distal segments, dyf-18 mutants can build a full-length cilium. Altogether, my studies offer insights into functional aspects of two novel proteins required for the maintenance of ciliary function in C. elegans.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Michel Leroux
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Crystallographic analysis of birnavirus VP4 proteases.

Date created: 
2012-12-12
Abstract: 

Birnaviruses have a bi-segmented double-stranded RNA genome residing within a single-shelled non-enveloped icosahedral particle. There are economic reasons to study birnaviruses and prevent their propagation as many of the birnaviruses are pathogenic to species that are consumed by humans (salmon, yellowtail fish, chicken, and clam) and some are commercially farmed. Protease VP4 cleaves the polyprotein (NH2-pVP2-VP4-VP3-COOH) of birnavirus into components required for virion assembly. It utilizes a serine-lysine (Ser/Lys) catalytic dyad that is less characterized than the Ser/His/Asp classical catalytic triad; thus, there are also scientific interests in studying its mechanism. The crystal structure of Infectious pancreatic necrosis virus (IPNV) revealed acyl-enzyme complexes suggesting that VP4 proteases could be used to trap different stages of the reaction mechanism. Here, I present the results of the crystallography analysis on VP4 proteases from Tellina virus 1 (TV-1), and Yellowtail ascites virus (YAV). These structures provided insights on how VP4 proteases interact with the substrates and how the polyprotein is cleaved; thus, will aid in the design of anti- birnavirus compounds. TV-1 was first isolated from the sand dwelling marine bivalve mollusk Tellina tenuis (clam). Manifestations of the disease include a thinner and chalkier shell as well as a pale yellow digestive gland. The structure of TV-1 VP4 was solved to 2.1 Å resolution revealing an intramolecular (cis) acyl-enzyme complex which demonstrates how the enzyme recognizes its own carboxy-terminus during the VP4/VP3 cleavage event. To our knowledge, this is the first time that an intramolecular acyl-enzyme has been observed within a protease crystal structure. YAV infection leads to ascites in yellowtail fish (Seriola quinqueradiata), which is popular in sushi. The existence of a previously proposed internal cleavage site within VP4 was confirmed using protein and fluorometric peptide cleavage assays as well as capturing it in two acyl-enzyme structures. The native active site structure (2.5 Å resolution) revealed both the acyl-enzyme and product bound states. The lysine mutant structure (2.3 Å resolution) revealed the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Dr. Mark Paetzel
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Crystallographic analysis of Bacillus subtilis signal peptide peptidase (SppA)

Author: 
Date created: 
2012-12-06
Abstract: 

Secreted proteins are initially synthesized in a precursor form that contains an N-terminal signal peptide for targeting the proteins to the cytoplasmic membrane. Upon translocation across the membrane via the Sec machinery, the signal peptide is cleaved off by signal peptidase. The remnant membrane embedded signal peptide is then cleaved within its hydrophobic core by signal peptide peptidase A (SppA). SppAs are membrane bound peptidases found in archaea, plant chloroplasts and bacteria. SppAs utilize a serine nucleophile and a lysine general base as catalytic residues. My PhD research has focused on the Bacillus subtilis SppA (SppABS). During my PhD study, I solved the three-dimensional structure of SppABS using X-ray crystallography, allowing us to study the difference and similarities between Gram-positive SppABS and the previously solved Gram-negative Escherichia coli SppA (SppAEC). Both proteins form similarly sized dome-shaped multi-subunit structures where the catalytic residues reside inside the concave portion of the dome. However, each subunit of SppABS is half the size of the SppAEC subunit, thus SppABS is an octameric complex while SppAEC is a tetramer. The structure revealed eight active sites in SppABS where three of the protomers come together to form one complete active site whereas SppAEC contains only four active sites. Structural analysis on the substrate binding pockets S1 and S3, along with activity assays using a range of peptide substrates, revealed that SppABS prefers substrates with leucine, arginine or tyrosine at the P1 position – a significantly different profile from SppAEC which prefers leucine but not arginine or tyrosine at the P1 position. I also solved the crystal structure of SppABS in complex with its own C-terminus bound within its active site. This structure has provided information on how the enzyme recognizes its substrates, confirms our previous SppABS substrate preference analysis and has posed a new question “Why does SppABS cleave its own C-terminus?” We show that SppABS cleaves its own C-terminus in an intra-complex fashion and mutational analysis shows that Tyr331 is important for self-cleavage. I also showed that SppABS is able to digest folded proteins which suggests that SppABS possibly has a membrane quality control function.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Mark Paetzel
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Investigations of micromonospora viridifaciens mutant sialidases in transglycosylations and novel enzymatic activity

Date created: 
2013-12-05
Abstract: 

Sialic acid is a monosaccharide with a nine-carbon atom backbone and there are more than fifty known derivatives of sialic acid depending on the substitution pattern, and these compounds make up the sialic acid family. The most widespread sialic acid family members are N-acetylneuraminic acid, N-glycolylneuraminic acid and Kdn. Sialic acid family members usually reside at the termini of sugar branch of glycoproteins and glycolipids in animals. As a result, the location of these sugars contributes to their roles in cellular and molecular recognition events. The present thesis focused on the biological mask function of sialic acid in the clearance and regulation of serum glycoconjugate lifetimes. Attaching sialic acid directly to terminal sugar residues can block the underlining galactose residues from recognition by the Ashwell receptors and tailor the glycoproteins clearance rate. Therefore, if sialic acid can be directly attached to recombinant glycoprotein drugs this will potentially decrease glycoprotein clearance rates; thereby allowing the glycoprotein drug’s dosage to be reduced. The present thesis focused on studying the ability of mutant Micromonospora viridifaciens sialidases to transfer sialic acid onto other sugar acceptors. The wild-type sialidase favors hydrolysis rather than transfer of sialic acid. We show that the Y370G mutant sialidase is flexible in transferring sialic acid onto several different sugar acceptors. To try and improve the yield for the transglycosylation reaction of the Y370G mutant sialidase, hydrophobic loops, which were incorporated on the enzyme to try and increase the local concentration of acceptor. The resultant loop mutants abolished the enzyme’s activity. Therefore, we targeted mutagenesis of the loop region to create a library of Y370G-loop mutants. Unfortunately, screening of this mutant library did not yield any promising next generation Y370G mutants. We also screened another mutant library based on two tyr370 nucleophile mutants, histidine (Y370H) and methionine (Y370M), with the goal of finding novel mechanisms of action. However, screening of this library produced several revertant mutants, but no promising candidates with either histidine or methionine as the enzymatic nucleophile.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Andrew Bennet
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

The curvature sensitivity of membrane-binding amphipathic helices can be modulated by the electrostatics of their flanking regions

Date created: 
2013-12-05
Abstract: 

Membrane-induced amphipathic helices (m-AH) can act as membrane curvature sensors by binding preferentially to hydrophobic lipid packing defects enriched in curved surfaces. Weak electrostatic interactions can impart a greater reliance on hydrophobicity and membrane curvature for binding. I probed the role of modifying membrane and protein charge on the curvature sensing of two m-AH containing proteins, CTP:phosphocholine cytidylyltransferase (CCT) and alpha-synuclein (alpha-syn). The m-AH in both proteins are flanked by disordered tails with multiple phosphoserines (CCT) or acidic residues (alpha-syn), which I mutated to glutamate or serine to modify protein charge. Analysis of binding to vesicles of varying curvature showed that increasing negative charge of the tail region decreased binding strength and augmented curvature dependence, which I attribute to charge repulsion. Conversely, increasing the membrane negative charge dampened the curvature dependence. Our data show that discrimination of curved vs. flat membranes with high negative charge could be modulated by phosphorylation.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Dr. Rosemary B. Cornell
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.