Canonical Wnt, or Wingless (Wg) in Drosophila, is an evolutionarily well-conserved signalling pathway that is important for a wide range of processes, including cell fate determination, axis formation and stem cell renewal. Wg signalling primarily functions to regulate the cytosolic stability of the key effector β-catenin (Armadillo, Arm, in Drosophila). Arm promotes the transcription of Wg target genes but also is required for the formation of stable adherens junctions. Previously, the Verheyen lab identified the non-muscle myosin II regulator Myosin Light Chain Phosphatase (MLCP) as a putative regulator of Wg signalling. Here we find that reducing the expression MLCP components leads to the attenuation of Wg target gene expression. I present our evidence that MLCP knock down directly regulates Wg signal transduction and that this regulation is through Arm localization. Thus, our work supports mounting evidence of a regulatory relationship between the adherens junctions and the Wg signalling pathway.
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to rhythmic oscillations in the heart and brain. Upon membrane hyperpolarization, HCN channel pore opening is coupled to inward movement of the S4 helix within the transmembrane voltage sensing domain (VSD, helices S1-S4). The gating pathway is proposed to include an initial voltage-dependent VSD movement step followed by a voltage-independent pore movement step (a cyclic allosteric mechanism). Various other mechanisms influence open state stability: A cytosolic cyclic nucleotide-binding (CNB) fold destabilizes the open state when unliganded (an autoinhibition mechanism), whereas binding of the phospholipid PIP2 to the transmembrane domain stabilizes the open state. After pore opening, the channel undergoes a mode-shift, presumed to include lateral movement of S4 towards S2, forming a more stable open state. Despite the knowledge of open state stabilization mechanisms, it remains unclear how these mechanisms affect the kinetics of the gating pathway. Do these mechanisms apply equally strongly to channel thermodynamics and kinetics? Do they apply under a variety of cellular conditions? And do they regulate the VSD movement step, the pore movement step, or both? In this work I examined both the thermodynamics and kinetics of the activation and deactivation pathways in a variety of HCN channel derivatives. I used two-electrode voltage clamp to determine that while channel thermodynamics follow the predictions of the autoinhibition model, a channel with an unliganded CNB fold has faster activation than a channel with autoinhibition relieved by CNB fold deletion. I propose this fast activation is promoted by a “quickening conformation” of the intact CNB fold. The quickening conformation is independent of PIP2 in both autoinhibited and autoinhibition-free channels. I used voltage clamp fluorometry to determine the speed of a VSD movement during channel deactivation in relation to pore closure. The speed of this VSD movement did not limit the rate of the deactivation pathway at strong depolarizations and showed stronger voltage dependence than pore closure. The speed of this VSD movement was independent of both cAMP binding and mode shift. Together my results clarify the HCN gating mechanisms of cyclic allostery, autoinhibition, PIP2 potentiation and mode shift, and produce novel models of both HCN channel activation and deactivation.
For over 30 years, researchers have taken advantage of genetic balancers and forward genetic screens to isolate lethal mutations, which have been studied to identify essential genes in C. elegans. Using traditional genetic methods, such as genetic mapping, complementation tests, and transgenic rescue assays, many essential genes have been successfully identified. However, to pinpoint a specific essential gene the involved experiments are usually labor intensive and time consuming. Nowadays, genetic methods combined with whole genome sequencing (WGS) and bioinformatics analysis provide an effective approach for the molecular identification of essential genes. In my thesis I successfully identified 64 new essential genes with 107 lethal mutations in genomic regions of C. elegans of around 14 Mb from Chromosome III(mid) and Chromosome V(left), by combining genetic mapping, Illumina sequencing, bioinformatics analyses, and experimental validation. Most of these genes have multiple recovered mutant alleles. Of these 64 genes 5 have new alleles identified, which had not been previously studied by RNA interference depletion. Furthermore, by investigating the locations of lethal missense mutations in essential genes, I have identified five novel protein functional domains. Functional characterization of the identified essential genes shows that most of them are enzymes, including helicases, tRNA synthetase, and kinases. There are also ribosomal proteins. Gene Ontology functional annotation also indicates that essential genes tend to execute enzyme and nucleic acid binding activities during fundamental processes, such as intracellular protein synthesis. Essential gene analysis shows that compared to non-essential genes, essential genes have fewer paralogs, and encode proteins that are in protein interaction hubs. Essential genes are also more abundantly and consistently expressed over all developmental stages than non-essential genes. All these essential genes traits in C. elegans are consistent with those of human disease genes. Unsurprisingly, most (90%) human orthologs of essential genes in this study are related to human diseases. Therefore, functional characterization of essential genes underlines their importance as proxies for understanding the biological functions of human disease genes.
Signal transduction pathways are crucial for co-ordinated development and growth of multicellular organisms. Dysregulation and mutations of components in these pathways can often lead to tumourigenesis. Evolutionarily conserved Homeodomain-Interacting-Protein-Kinase (Hipk) is a strong growth regulator of many signal transduction pathways, and elevated levels of hipk lead to tumour-like masses. While many known regulators of Hipk exist, we attempted to identify novel phospho-regulators of Hipk activity. Here we present evidence that Hopscotch, a core tyrosine kinase of the JAK/STAT cascade, is a putative phospho-regulator of Hipk activity in multiple contexts. We show that modulation of Hipk expression levels modifies JAK/STAT activity in both wildtype and tumourous tissues. Finally, we show that Hipk interacts with the JAK/STAT transcriptional effector STAT92E. Thus, our work provides a role for Drosophila Hipk in tumourigenesis and regulation of the JAK/STAT cascade.
Cilia are highly-conserved organelles ubiquitously present in metazoans and some unicellular eukaryotes. In humans, ciliary defects result in a plethora of serious genetic diseases termed ciliopathies. Despite their diverse morphology and function, cilia are comprised of a core set of proteins, and many ciliary genes share similar but likely not identical regulation mechanisms. Our research aims to understand the variations in cis-regulatory elements in ciliary genes and the impact of such variations on transcriptional regulation. We hypothesize that cis-regulatory elements in different ciliary promoters are unique and that this uniqueness impacts the expression and function of ciliary genes. We focus on a particular cis-regulatory element, the X-box motif, which functions as the binding motif for RFX/DAF-19, a transcription factor that regulates ciliary gene expression. We identify and analyze X-box motifs for a set of 32 well-studied ciliary genes in C. elegans and their orthologs in 25 additional nematodes, including both free-living and parasitic species. My research consists of three modules. First, we curate ciliary gene orthologs using a combined approach, including homology-based gene finding and RNA-seq-based improvement. The primary goal of this step is to ensure that the 5' ends of the genes are accurately defined in order to properly locate ciliary promoters. Second, we search for putative X-box motifs in these promoters using computational tools to identify motifs that resemble the consensus. For the promoters from which consensus X-box motifs are not found, we will search for X-box motifs that may show more differences from the consensus using frequency matrix-based search and regular expressions, which we call "atypical" X-box motifs. Third, we analyze the putative atypical X-box motifs, focusing on their sequence similarities, positions in promoter sequences, and flanking sequences, and compare them against the consensus X-box motifs. In this study, I will highlight progress made and challenges encountered for defining X-box motifs in ciliary genes.
Short-read DNA sequencing technologies have revolutionized bacterial genomics, but these technologies have limitations. It is easy to produce a high quality draft genome, but relatively costly and/or time consuming to complete a genome, so most bacterial genomes remain as drafts. Despite this, limitations of draft bacterial genomes for functional analysis have not been well assessed. To characterize the importance of missing and poor quality regions of draft genomes, analyses of COG categories and genes of medical importance were performed using analogous draft and complete genomes. A popular genomic island prediction tool, IslandViewer, was updated to allow draft genomes as input, and its ability to detect genomic islands in draft genomes was assessed. There are limitations to bacterial draft genome analysis, with respect to disproportionately missing certain types of genes. However, valuable information of medical interest, including virulence and antimicrobial resistance genes, can still be obtained from some draft genome datasets.
Advances in whole genome sequencing (WGS) technologies have created an era in which WGS can routinely be integrated into disease outbreak investigations for the rapid detection and characterization of the causative agents. Although most genomic investigations of outbreaks to date focus on using single nucleotide variations to help track the spread of disease, this dissertation focuses on efforts to improve the characterization of large clusters of horizontally-acquired genes, named genomic islands (GIs), that may cause large phenotypic changes. Such mobile elements contribute a fundamental role in the rapid adaptation of microbial life to various changes in the environment and are known to encode genes involved virulence, antimicrobial resistance (AMR) and alternative metabolism. I present the integration of rich gene annotations of virulence factors (VFs), AMR genes, and pathogen-associated genes into IslandViewer, a web server for the prediction of GIs in addition to the re-design of the web server to now include an interactive genome visualization library named GenomeD3Plot. I also present the application of IslandViewer for GI analysis on real outbreak data from multiple Listeria monocytogenes food-borne outbreaks from across Canada to show that isolates from geographically and temporally distinct outbreaks have unique sets of GIs. In addition, I present an analysis coupling the rich AMR gene annotations with GI predictions over a large collection of diverse microbial genera that revealed AMR genes as a whole are not over-represented within GIs, in contrast to VFs as have been previously studied. However, upon breaking down the dataset, certain classes of resistance were found to be associated with such mobile regions. Lastly, I present a WGS study of L. monocytogenes to elucidate the contribution of genetic changes to the ability of this pathogen to tolerate and grow in harsh environments, especially cold temperatures, that are important for its role in causing disease. Overall, this work contributes to improved characterization of GIs as well as a better understanding of trends in the role of GIs and mobile regions in the context of AMR and infectious disease.
Development from a single cell into a multicellular entity is controlled by the intricate regulation of different cell signalling molecules. Wnt/Wingless (Wg) is one such evolutionarily conserved molecule that plays a critical role in cell fate specification, tissue patterning and organ development. Aberrant signalling leads to many developmental defects and cancer. The Wg pathway is regulated by reversible phosphorylation both in its silent and active states. Although several studies have shown the role of various kinases and phosphatases in regulating distinct steps of the Wg pathway, the entire cascade of events that regulate the pathway still remains elusive. To identify novel regulators of the Wg pathway, we performed an in vivo RNAi screen in the wing disc of Drosophila. This screen identified several new kinase and phosphatase modulators of the Wg pathway. Further characterization uncovered two proteins, the endosomal protein Myopic (Mop) and the serine threonine phosphatase Protein Phosphatase 4 (PP4), which are essential for Wg pathway activity. Knockdown of mop caused Wg protein accumulation in both the Wg secreting and receiving cells. Loss of Mop caused reduced Wg secretion due to the accumulation of Wg and Wntless in endosomes of secreting cells. The defective secretion and aberrant accumulation of Wg was rescued by overexpression of the endosomal protein Hrs. The vertebrate homolog of Mop, HDPTP has similar roles in regulating Wnt trafficking in mammalian cells. In Wg receiving cells, mop knockdown causes accumulation of the Frizzled receptor in early endosomes. Loss of hrs phenocopies this effect on Fz. Histochemical and genetic analyses suggested that Mop protects Hrs from lysosomal degradation by both promoting its deubiquitination by Ubpy and inhibiting the ubiquitin ligase Cbl. Thus, Mop stabilises Hrs in the endosomes, which promotes trafficking of Wg pathway components both in the signal sending and receiving cells. My work provides useful insight on how Mop-Hrs-Ubpy regulates the endosomal trafficking and signalling output of the Wg pathway. The serine threonine phosphatase PP4 also plays an important role in the Wg pathway. PP4 influences Notch pathway-driven wg transcription. Knockdown of PP4 affects expression of Notch pathway components and impairs growth of Drosophila appendages. These defects were rescued by the overexpression of nuclear Notch. Together, these studies provide the first evidence implicating a role for Mop and PP4 in trafficking and transcription of Wg.
Type I signal peptidase, SPase I, is an essential bacterial enzyme participating in the process of protein secretion. SPase I catalyzes the conversion of pre-proteins to mature proteins by cleaving off the amino-terminal signal peptides from the pre-proteins during protein secretion. The removal of these remnant signal peptides, required for the continuation of the secretion process, is not a well understood process in bacteria. In Escherichia coli, signal peptide peptidase A, SppA, together with other enzymes, is responsible for the removal of these remnant signal peptides. This thesis work focuses on characterizing and comparing Staphylococcus aureus SPase I, SpsB, with other bacterial SPase I as well as characterizing a previously unexamined SppA related enzyme, E. coli signal peptide peptidase A2, SppA2. A fluorescent lipidated peptide substrate with a Gram-positive signal peptide sequence was used to characterize the Michaelis-Menten kinetic constants for S. aureus SpsB along with SPase I from Bacillus subtilis, Staphylococcus epidermidis, and E. coli. E. coli SPase I has a significantly lower catalytic efficiency towards the substrate. A previously characterized E. coli pre-protein was mutated to match the sequence of the Gram-positive peptide sequence, leading to a significantly reduced maturation rate by E. coli SPase I, both in vitro and in vivo. These results have led to the discovery of a previously uncharacterized residue in the SPase I substrate binding groove, proline 88 in E. coli, which may contribute to the difference in catalytic efficiency observed between Gram-positive and Gram-negative SPase I enzymes. Limited proteolysis has revealed that E. coli SppA2 has an N-terminal protease sensitive region, residues 39 to 91, and a C-terminal trypsin resistant ,trSppA2, domain, residues 92 to 349. Light scattering results indicate that trSppA2 forms octamers in solution with a proposed dome-shape structure similar to that of E. coli and B. subtilis SppA. Activity and mutagenesis studies demonstrate that trSppA2 can digest both small peptides and folded proteins, with a preference for hydrophobic substrates, while the S178A and K230A mutants are inactive suggesting that SppA2 is a serine / lysine dyad enzyme. Lastly, the protease sensitive region is required for proper protein folding and may regulate substrate traffic into and out of the inner cavity of the SppA2 octamer.
Gram-positive Bacillus subtilis (B. subtilis) signal peptidase I (SPase I) is a membrane-bound endopeptidase that cleaves off the amino-terminal signal peptide from pre-proteins before or after their translocation across the cytoplasmic membrane. B. subtilis has five chromosomal SPases I; SipS, SipT, SipU, SipV, SipW, and two plasmid encoded paralogous SipP. SipS is one of major SPases I in the species which is essential for cell viability. It is also one of the closest of the B. subtilis SPase I enzymes in sequence to the well characterized Gram-negative E. coli SPase I. As a result, SipS was chosen for this research study. B. subtilis SipS uses Ser/Lys catalytic dyad for catalytic activity, utilizing Ser43 and Lys83 in the enzyme. The constructs - SipS Full-Length (FL), SipS ∆2-35 Wild-Type (WT), SipS ∆2-35 S43A and SipS ∆2-35 K83A – were expressed, purified, and screened for crystallization conditions. Catalytically active SipS ∆2-35 WT formed needle shaped crystal clusters whereas SipS ∆2-35 K83A produced initial hits in crystallization conditions containing lithium sulfate. Preliminary data for the catalytic activity of B. subtilis SipS ∆2-35 WT shows that hexaaminecobalt (III) chloride inhibits the enzyme.