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

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Structural and kinetic analysis of Escherichia coli signal peptide peptidase A

Date created: 
2013-06-19
Abstract: 

Secretory proteins contain a signal peptide at their N-terminus. The signal peptide functions to guide proteins to the membrane and is cleaved off by signal peptidase. The remnant signal peptides must be removed from the membrane to prevent their accumulation which can lead to membrane destabilization. Escherichia coli signal peptide peptidase A (SppAEC) has been identified as a major enzyme that processes the remnant signal peptide to smaller fragments. SppAEC, however, had remained uncharacterized such that the structure, catalytic mechanism, and substrate preference were unknown. I have cloned, overexpressed, purified and crystallized an active soluble domain of SppAEC. We have determined the structure of SppAEC by X-ray crystallography, which revealed that: 1) SppAEC has two similar alpha-beta domains (despite limited sequence identity), 2) SppAEC forms a tetramer that results in a dome shaped structure with a hydrophobic interior and hydrophilic exterior, 3) the four active sites within the cavity each utilize a Ser-Lys dyad where the general base (Lys209) arrives from the N-terminal domain and the nucleophile (Ser409) from the C-terminal domain. I have further characterized SppAEC using steady-state kinetics and cocrystallization. Using a series of fluorometric peptide substrates, I discovered that leucine is the most preferred residue at the P1 substrate position. I cocrystallized an SppAEC active site mutant (K209A), in complex with the substrate Z-LLL-MCA. The electron density within the active site of the 1.95 Å resolution structure is consistent with the carbonyl carbon of the substrate’s C-terminal leucine being covalently linked via an ester bond to the Ser409 O, thus revealing an acyl-enzyme complex in SppAEC. This is direct evidence that Ser409 O serves as a nucleophile in the SppAEC catalyzed reaction and confirms the identity of the S1 and S3 substrate specificity pockets. Lastly, I measured the pH dependence of both WT and S431A enzymes (Ser431 Ois hydrogen-bonded to Lys209 Nζ). The pH-rate profiles are consistent with S431 playing a role in lowering the pKa of the lysine general base, which would be critical for activity at physiological pH. We propose that the active site architecture of SppAEC may be best described as a Ser-Lys-Ser triad.

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

Refolding Recombinant Escherichia coli BamA: the integral membrane component of the BAM (Beta-barrel Assembly Machinery) complex

Author: 
Date created: 
2014-07-23
Abstract: 

The selective permeability of the outer membrane (OM) of Gram-negative bacteria is mainly determined by the pore-forming proteins within it, which are called outer membrane proteins (OMPs). The proteinaceous apparatus required to fold and assemble the OMPs is known as the beta-barrel assembly machinery (BAM) complex. In Escherichia coli, the BAM complex is made up of five proteins. BamA is a beta-barrel protein integrated into the OM, while BamB, BamC, BamD and BamE are lipoproteins attached to the periplasmic side of the OM by a covalently bound lipid. Over-expression of BamA without its signal peptide results in insoluble inclusion body formation. I have investigated refolding strategies for E. coli BamA. The refolded BamA has been analysed by size-exclusive chromatography, heat-modifiability, circular dichroism spectroscopy and limited proteolysis. The experimental results are consistent with both a full length BamA and a truncated BamA construct being refolded properly using my discovered conditions. Both refolded constructs are able to crystallize. These initial crystallization conditions will be helpful in the structural analysis of E. coli BamA.

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

The functional characterization of the gene coding for O-linked β-N-acetylglucosaminidase (OGA) in Drosophila melanogaster

Date created: 
2014-08-15
Abstract: 

O-linked β-N-acetylglucosaminidase (OGA) is the enzyme responsible for removing the O-linked β-N-acetylglucosamine (O-GlcNAc) modification from serine and threonine residues of a variety of proteins, while its addition to protein targets is catalyzed by O-linked β-N-acetylglucosamine transferase (OGT). sxc/Ogt is essential in Drosophila melanogaster; however, it is unknown whether Oga is also essential in flies. I found that, in flies, a significant decrease in Oga transcript induced by RNAi knockdown is not lethal and that a nonsense mutation that putatively results in the translation of a C-terminally truncated version of OGA is viable when crossed to a deficiency known to span the Oga locus in the genome; however, reduced viability was observed when ubiquitously overexpressing two copies of Oga cDNA. Reduced expression of Oga and Ogt in Drosophila insulin-producing cells, via targeted RNAi expression with a dILP2-GAL4 driver, results in a slight increase and decrease, respectively, in male body weight.

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

A search for new players in the signalling pathways regulating dorsal closure of the Drosophila embryo

Author: 
Date created: 
2014-08-14
Abstract: 

Dorsal closure in Drosophila is a widely used model system to study developmental epithelial fusions and wound healing, and had been shown to require coordinated cell morphogenesis and reciprocal communication between different tissue types. In this thesis, I focused on identifying new components of the signalling pathways regulating dorsal closure. First, I extended the characterization of Epidermal Growth Factor Receptor (EGFR) signalling, which is an important regulator of dorsal closure, and established new leads in the search for components upstream and downstream of EGFR during dorsal closure. I have also identified the ligand Folded gastrulation (Fog) as a candidate upstream regulator of signalling during dorsal closure. Fog appears to regulate the transcript levels of decapentaplegic and zipper, two important genes known to participate in dorsal closure. Finally, I initiated studies to explore if regulators of cell adhesion at the neuromuscular junction are conserved regulators of cell adhesion during dorsal closure.

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

Transcriptional regulation of the ciliopathy gene MKS1/mks-1

Author: 
Date created: 
2014-06-12
Abstract: 

Meckel Grubber Syndrome (MKS) is a severe ciliopathy. The first identified causal loci of MKS resided in the gene MKS1. The only known transcription factor of MKS1 is Regulatory Factor X (RFX). C. elegans, an organism with well-characterized ciliated neurons, is ideal for studying ciliary genes. Many ciliary genes including MKS1 are conserved in C. elegans. My study aims to find candidate mutants of transcription factor(s) for mks-1, the C. elegans ortholog of MKS1. In order to track in vivo mks-1 expression in C. elegans, I generated a transgenic strain that expressed Green Fluorescence Protein (GFP) driven by mks-1 promoter. I carried out genetic screens to identify mutations that altered GFP fluorescence intensity and expression profile, as a way to search for potential transcription factor(s) for mks-1 in C. elegans. I successfully found an X-linked recessive mutant, which suppresses mks-1 reporter gene expression in subset of labial neurons. The mutant has normal labial neurons development. This research has set up a solid stage for studying the transcriptional regulation of mks-1 and other ciliary genes.

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

Investigation into the catalytic role of threonine 712 in VP4 protease from the blotched snakehead virus

Author: 
Date created: 
2014-07-24
Abstract: 

The blotched snakehead virus (BSNV) is a member of the Birnaviridae family; it is characterized by a single-shelled capsid and a bisegmented, double-stranded RNA genome. One segment of its genome, segment A, encodes the BSNV polyprotein, NH3+-pVP2-X-VP4-VP3-COO-, which includes VP4 - a peptidase that uses a 'nonclassical', Ser-Lys catalytic dyad mechanism. VP4 cleaves the polyprotein at specific recognition sites to release four peptides and four polypeptides. The polypeptides include the capsid protein, VP2, protein X (whose function is unknown), the multifunctional protein VP3, and also VP4. Previous site-directed mutagenesis and crystallographic studies of BSNV VP4 have identified the serine nucleophile and lysine general base, however the potential roles played by other residues within the active site have yet to be experimentally investigated. One conserved active site residue of interest is Thr712, which is appropriately positioned, according to the BSNV VP4 crystal structure (PDB ID 2GEF), to form a hydrogen bond with the active site's general base, Lys729. Herein, we present data supporting Thr712's significance to VP4-mediated catalysis from experiments using site-directed mutagenesis and time-course cleavage assays. We also describe the crystallization of a truncated, active site mutant construct - K729A - of BSNV VP4.

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

Characterization of Molecular Correlates of the Chronic Humoral Immune Response: Clues towards Eliciting Broadly Neutralizing Antibodies against HIV

Date created: 
2014-08-07
Abstract: 

A central focus in the pursuit of an effective vaccine against HIV-1 is the induction of broadly neutralizing (bNt) antibodies (Abs). However, these Abs are rarely elicited in natural infection and the immunological processes that drive this occurrence are not clearly defined. Modulation of the immunological environment during the chronic phase of infection provides a plausible mechanism by which bNt Abs are elicited and also advances the possibility that vaccine design should account for the immune response specific to this phase. Thus, the work presented here seeks to characterize the chronic phase of the humoral immune response to gain insight into the immunological mechanisms that generate bNt-Ab responses. First, disturbances in B-cell distribution in HIV+ individuals were evaluated using a computational algorithm, flowType, compared to a manual-analysis tool. In addition to yielding similar results to manual analysis, flowType revealed dysregulation of novel B-cell subsets in HIV, providing a representation of the global extent of HIV-associated B-cell dysregulation. Second, changes in B-cell distribution and serum reactivity were compared between HIV+ individuals who followed different clinical courses and individuals with the autoimmune disease, systemic lupus erythematosus (SLE). B-cell dysregulation was evident in both cohorts, though more extensive in HIV. Sera from HIV+ individuals with increased viral load, as well as SLE patients, displayed significant polyreactivity as compared to HIV+ sera from those controlling their infections. These results illustrate that despite similarities in cellular perturbations, the Ab response against HIV is antigen driven rather than autoimmune. In addition, immunogenetic analyses of B-cell repertoires of HIV+ individuals demonstrated perturbations within antigen-inexperienced naïve B cells as well as increased somatic-mutation levels, which were observed in the effector B-cell subpopulations. Finally, a murine model for chronic infection was developed using immunization with filamentous bacteriophage as a model virus-like particle. Functional analyses of CD138+ plasma cells showed that a single immunization elicited innate-like and type-1 T-cell independent (TI-1) B-cell responses, and prolonged immunization resulted in perturbations reminiscent of those observed in chronic infections (i.e., plasma-cell exhaustion). Preliminary data from transcriptomic analyses of plasma cells from different time points after immunization suggests immunophenotypic differences within the population, indicating either developmental changes, or selection from different precursor populations. Collectively, the data provide deeper insight into changes occurring in short-term and chronic humoral immune responses, as well as establishing a functional animal model that may be used to probe these features further.

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

Bidirectional communication between tissues regulating morphogenesis in a Drosophila model of wound healing

Author: 
Date created: 
2014-05-28
Abstract: 

Drosophila embryonic dorsal closure is a developmental epithelial fusion event that resembles vertebrate processes such as neural tube closure and wound healing. Dorsal closure involves the migration of two epidermal sheets to close an epidermal hole occupied by a tissue called the amnioserosa. The successful completion of this process requires a signalling network that involves communication between the amnioserosa and the dorsal epidermis, and the Dpp and JNK pathways are two central participants. Dorsal closure is driven in part by myosin-dependent cell shape change in both the dorsal epidermis and the amnioserosa. Consistent with this, myosin heavy chain expression from the zipper (zip) gene is upregulated in these two tissues in a Dpp-dependent manner and we believe that zip regulation is a critical component of dorsal closure. We have taken a candidate gene approach to identify new participants in zip regulation during dorsal closure. We have established that Egfr acts as a brake on dorsal closure in that it inhibits the expression of both dpp and zip. Egfr is itself negatively regulated by the non-receptor tyrosine kinase Ack, which may be reducing Egfr levels by promoting its endocytosis. Previous work suggests that a diffusible signal from the amnioserosa regulates gene expression in both the amnioserosa and the dorsal epidermis. We have established that this diffusible signal is the steroid hormone ecdysone, which cooperates with JNK signalling to turn on zip expression. We have also determined that Dpp secreted from the dorsal epidermis turns on ecdysone production in the amnioserosa by promoting expression of the ecdysone biosynthetic enzyme Spook. Thus, we have uncovered reciprocal signalling between two tissues leading to coordinated morphogenesis. We have further established that ecdysone is acting in a non-canonical manner in its cooperation with JNK signalling in this process. We provide genetic and immunohistochemical evidence that the ecdysone receptor turns on zip by complexing with the JNK-activated transcription factor AP-1. Using the proximity ligation assay, we visualize this complex in the nuclei of amnioserosa and dorsal epidermal cells during dorsal closure. Our findings may provide clues to the signalling events occurring during other epithelial fusions.What we found in this project could help to better understand the bidirectional communication that occurs between the amnioserosa and dorsal epidermis at the level of signaling network allowing coordinated morphogenesis of the two tissues through the regulation of actomyosin contractility.

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

Structural analysis of human cardiac troponin C and myosin binding protein C

Date created: 
2014-07-25
Abstract: 

Sarcomeric proteins are essential for the proper structural assembly and functioning of the sarcomere, the basic contractile unit in striated muscles. When mutations are present in the genes that encode for these proteins, it may lead to cardiac diseases, such as hypertrophic cardiomyopathy (HCM), the leading cause of death in young athletes. Hundreds of mutations within the genes that encode sarcomeric proteins have been shown to cause HCM. The L29Q mutation in cardiac troponin C (cTnC) and the R502W mutation in cardiac myosin binding protein C (cMyBP-C) are two of these mutations. cTnC senses the cytosolic Ca2+ concentration and transduces this signal to allow for cross-bridging, leading to muscle contraction. cMyBP-C regulates muscle contractility by interacting with myosin and actin. How the L29Q and the R502W mutations, respectively, affect the cTnC and cMyBP-C and cause disease is unclear. To advance our knowledge of how these two mutations affect the cardiac proteins’ structures and functions, I first generated high resolution structures of the wild type (WT) and mutant regulatory domains of cTnC (cNTnC) using X-ray crystallography and then, determined the WT and R502W mutant structures of the C3 domain of cMyBP-C using nuclear magnetic resonance (NMR). The WT cNTnC was discovered to have coordinating Cd2+ ions at both of its calcium-binding sites. This is true for the mutant cNTnC as well. In the WT cNTnC, the vestigial site (EF1) coordinated Cd2+ in a noncanonical ‘distorted’ octahedral geometry, while the functional calcium-binding site (EF2) coordinated Cd2+ in the canonical pentagonal bipyramidal geometry. A subtle structural change was observed in the region near the L29Q mutation, and it may play a role in the increased Ca2+ affinity of the mutant. The R502W mutation in cMyBP-C did not change the protein’s global structure. The dynamics and thermal stabilities of the protein were also not affected by the mutation, as shown by techniques such as amide 15N relaxation and circular dichroism spectroscopy. The mutation does, however, alter the surface charge on the C3 domain and, like other HCM-related mutations found within the same domain, may disrupt the protein’s interactions with other sarcomeric proteins, such as actin. The data acquired from this thesis project contributes to a better understanding of the structures of sarcomeric proteins and the pathophysiology of hypertrophic cardiomyopathy.

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

Further molecular and genetic analysis of upSET, the MLL5 orthologue in D. melanogaster

Author: 
Date created: 
2014-07-18
Abstract: 

Several histone methyltransferases are products of the MLL (Mixed Lineage Leukemia) family of genes. Although their mechanisms of action remain elusive, these genes are important because they are often deleted in patients with certain types of leukemia. In most cases, MLLs function catalytically via their SET domains, and their modifications of histones are important for chromatin structure and gene regulation. Here I report my analysis of the fly orthologue of MLL5, upSET. In contrast to an earlier study, RNAi inhibition of upSET gene expression did not result in female sterility but was instead lethal. Although UpSET has a SET domain, down-regulation of upSET does not affect global H3K4-methyl levels. Unlike human MLL5, the SET domain of UpSET does not appear to be an OGT substrate. Additionally, I have characterized UpSET as a suppressor of variegation; this suggests that UpSET is involved in establishment and/or maintenance of chromatin structure and accessibility.

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