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

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Transcription to Trafficking: Novel Regulation of Wnt/Wingless (Wg) Pathway by Protein Phosphatases

Date created: 
2015-03-03
Abstract: 

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.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Esther Verheyen
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

Biochemical characterization of signal peptide processing enzymes: Staphylococcus aureus signal peptidase I and Escherichia coli signal peptide peptidase A2

Date created: 
2015-04-13
Abstract: 

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.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Mark Paetzel
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

Crystallization of Bacillus subtilis Type I Signal Peptidase S (SipS)

Author: 
Date created: 
2015-03-30
Abstract: 

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.

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

The discovery of several novel deflagellation genes and the identification of ADF1

Author: 
Date created: 
2015-12-07
Abstract: 

Defective cilia, hair-like organelles that protrude from the cell membrane, cause diverse human diseases. The Quarmby lab previously used the unicellular alga, Chlamydomonas reinhardtii, to uncover three genes involved in the ciliary stress response known as deflagellation. They showed that defects in a related gene in humans underlie a severe form of juvenile onset polycystic kidney disease. We postulated that rare or subtle variants in additional disease genes could be uncovered by using a more sensitive genetic screen. Armed with advances in whole genome sequencing and an enhanced enrichment protocol, we recovered multiple alleles of previously isolated genes and several alleles of three novel deflagellation genes. We identify the previously elusive ADF1 as TRP15, a putative cation channel activated by intracellular acidification. We also identify the novel deflagellation genes ADF2 as a putative Ins(1,3,4)P3 5/6 Kinase, ADF5 as FAP16 and ADF4 as a glycosyltransferase exhibiting a novel starvation-specific deflagellation defect.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Lynne Quarmby
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Crystallization of Mycobacterium tuberculosis type I signal peptidase

Date created: 
2015-03-25
Abstract: 

Bacterial signal peptidase I (SPase I) plays a key role in the translocation of proteins across the cytoplasmic membrane. SPase I cleaves the signal peptide from the pre-protein. The unique Ser/Lys dyad mechanism, utilized by SPase I, is conserved in this family of enzymes in both Gram-positive and Gram-negative species. Mycobacterium tuberculosis (Mtb) SPase I is an attractive target for the design of antimicrobial compounds since it is essential for the bacterium’s viability and is involved in the translocation of important pathogenic proteins to their final destinations. Based on sequence alignments of bacterial SPase I, it is hypothesized that Lys174 and Ser96 are catalytically active residues and act as a general base and nucleophile in the Ser/Lys dyad, respectively. We have investigated the overexpression, purification and crystallization of three full length constructs including WT Mtb SPase I, Mtb SPase I K174A and Mtb SPase I S96A. After obtaining initial protein crystals of Mtb SPase I, different optimizing methods were applied to improve the crystal quality. The optimized crystal diffracted to 3.5 Å resolution. We present here analysis of the Mtb SPase I K174A crystals and initial data analysis.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Dr. Mark Paetzel
Department: 
Science:
Thesis type: 
(Thesis) M.Sc.

Phenotypic Analysis of NCR1, the Yeast Niemann Pick Type-C Disease Gene

Date created: 
2015-03-16
Abstract: 

In humans, the deadly neurodegenerative Niemann Pick Type-C disease (NPC) is caused by loss of function mutations in the NPC1 gene, which chiefly affect Purkinje neurons of the brain. While the underlying cause of NPC is believed to be lysosomal cholesterol accumulation, recent studies suggest that other lipid, protein and calcium defects contribute to the cellular defects in NPC. In contrast, deletion of the yeast NPC1 homologue, NCR1, does not cause any obvious growth or sterol trafficking defects. However, ncr1Δ cells are resistant to edelfosine, a phosphatidylcholine (PC) analogue drug that disrupts PC synthesis and distribution. Examination of PC trafficking in edelfosine treated ncr1Δ cells suggested that NCR1 deletion suppressed edelfosine-induced vacuolar PC trafficking defects. Ybt1p, the vacuolar membrane PC importer was found to be important for vacuolar PC uptake in edelfosine treated ncr1Δ cells. Because Ybt1p affects vacuolar calcium levels and human NPC neurons show calcium defects, ncr1Δ cells were tested for calcium phenotypes, where we found that these mutants were sensitive to high calcium concentrations. Both the ncr1∆-associated edelfosine and calcium defects were exacerbated in cells expressing multicopy LEU2, which encodes an enzyme required for leucine biosynthesis. Because leucine activates the vacuolar Target Of Rapamycin Complex 1 (TORC1), we investigated a potential link between Ncr1p and TORC1. Like ncr1Δ cells, several TORC1-associated mutants were edelfosine resistant and a mutation causing constitutive TORC1 activation exacerbated ncr1Δ edelfosine resistance. This work uncovered novel PC- and calcium-related defects in ncr1Δ yeast cells, which might provide insights into conserved functions of Ncr1p/NPC1 that relate to defects in NPC-affected Purkinje neurons. This phenotypic analysis also implicates TORC1 in some aspect of Ncr1p function in yeast.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Dr. Christopher T. Beh
Department: 
Science:
Thesis type: 
(Thesis) M.Sc.

Studies of RNA Processing and Localization: Diversity of small RNA in Plants and the Development of an in vivo RNA Imaging Tool.

Author: 
Date created: 
2016-01-22
Abstract: 

Eukaryotic genomes are extensively transcribed giving rise to thousands of non-coding RNAs, the biogenesis of which is highly conserved. While their functionality is debatable, evidence suggests that there are many RNAs, such as microRNAs and short interfering RNAs, involved in the regulation of gene expression. The processing of these RNAs together with their temporal and spatial expression patterns is, therefore, of central importance and requires the development of RNA imaging tools, which invariably rely on fluorescent microscopy. In this thesis I examine the expression of small RNAs (sRNAs) in the land plants and develop an RNA-based system for tracking and purification of cellular RNA complexes.To study the conservation of sRNAs and their biogenesis machinery across a broad spectrum of plants I conducted a survey of sRNA expression in 24 vascular plants. I found that conifers fail to produce a 24-nt class, which mediates heterochromatin formation in angiosperms, and instead produce a very diverse 21-nt size class, possibly generated by a novel Dicer-like (DCL) family that I discovered by searching conifer ESTs. I found no evidence of DCL3 – an enzyme responsible for the 24-nt size class production in angiosperms, indicating that conifers may utilize a diverse 21-nt class to help organize their unusually large genomes. Sequencing of sRNAs from a conifer P. contorta revealed many conserved miRNA families and other sRNAs, indicating that the sRNA-generating machinery was already present in the earliest spermatophytes. Since RNA lacks strong intrinsic fluorescence, it has proven challenging to track RNA molecules in real time. To address this problem I developed a new imaging method that relies on a high affinity RNA aptamer fluorophore system called RNA Mango. This aptamer binds to a series of Thiazole Orange derivatives with nanomolar affinities, while increasing their fluorescence up to 1100-fold. Imaging of RNA Mango by single-molecule fluorescence microscopy, together with visualization of RNA Mango-dye complex in C. elegans gonads demonstrates the potential for live-cell RNA imaging with this system. Furthermore, incorporation of RNA Mango into bacterial 6S RNA along with biotinylation of the fluorophore demonstrates that the aptamer can also be used for purifying biologically important RNAs.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Peter Unrau
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Regulation of the kinesin-3 motor, KIF1A, in a cellular model of Alzheimer’s disease

Author: 
Date created: 
2016-01-07
Abstract: 

Neurons are morphologically unique cells that rely on axonal transport for their function and viability. Amyloid-β oligomers (AβOs), a neurotoxin in Alzheimer’s disease (AD), disrupt axonal transport via dysregulation of signaling cascades. I assessed the role for glycogen synthase kinase 3β (GSK3β), a kinase implicated in AD, in the direct regulation of the kinesin KIF1A. Inhibition of GSK3β prevented transport defects in AβO-treated primary neurons, and co-immunoprecipitation studies confirmed an interaction between KIF1A and GSK3β. Mass spectrometry on KIF1A isolated from AD transgenic mouse brain (Tg2576) showed that within a regulatory domain, Ser 402 is phosphorylated and conforms to a GSK3β recognition site. The transport of a phospho-resistant (S402A) form of KIF1A was unaffected in AβO-treated neurons whereas KIF1A (S402E) transport is severely reduced. These data suggest that AβOs impair transport via GSK3β acting directly on KIF1A. Ultimately, this work may identify novel mechanisms of KIF1A regulation in AD.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Michael Silverman
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Dysregulation of neuronal calcium signaling impairs axonal transport independent of tau in a model of Alzheimer's disease

Date created: 
2014-10-30
Abstract: 

Neurons rely on microtubule-based, fast axonal transport of proteins and organelles for development, communication and survival. FAT impairment precedes overt cellular toxicity in multiple neurodegenerative diseases, including Alzheimer’s disease (AD). Intracellular Ca2+ dysregulation is also widely implicated in early AD pathogenesis; however, its role in transport impairment is unknown. Our lab was first to demonstrate that soluble amyloid-β oligomers (AβOs), proximal neurotoxins in AD, impair vesicular transport of axonal brain-derived neurotrophic factor (BDNF). Contrary to a central paradigm, I show that BDNF transport is blocked independent of the microtubule-associated protein, tau, microtubule destabilization, and acute cell death. Significantly, BDNF transport is impaired by non-excitotoxic activation of calcineurin (CaN), a Ca2+-dependent phosphatase. Based on these findings, I investigated Ca2+-dependent mechanisms that underlie the spatiotemporal progression of AβO-induced transport defects and dysregulate KIF1A, the primary kinesin motor required for BDNF transport. Because CaN and its effectors, protein phosphatase-1 (PP1) and glycogen synthase kinase 3β (GSK3β), are present in both dendrites and axons, I investigated if postsynaptic AβO binding impairs dendritic transport prior to FAT disruption. AβOs induce dendritic and axonal BDNF transport defects simultaneously; however, maximal dendritic transport defects are observed prior to maximal impairment of FAT. I correlated the spatiotemporal progression of transport defects with Ca2+ elevation and CaN activation in dendrites and subsequently in axons. Postsynaptic CaN activation converges on axonal Ca2+ dysregulation to impair FAT. Specifically, AβOs colocalize with axonal VGCCs, and blocking VGCCs prevents FAT defects. Finally, BDNF transport defects are prevented by dantrolene, a compound that reduces Ca2+-induced- Ca2+ release through ryanodine receptors in axonal and dendritic ER membranes. Together, these mechanisms activate CaN-PP1-GSK3β signaling and lead to inhibitory phosphorylation of KIF1A at a highly conserved consensus site within its dimerization domain. Collectively, this thesis establishes novel roles for Ca2+ dysregulation in BDNF transport disruption and tau-independent toxicity during early AD pathogenesis.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Michael Silverman
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

The ‘who, where and when’ of neurogenesis during Drosophila melanogaster eye development

Date created: 
2014-10-30
Abstract: 

The development of structurally diverse and functionally complex multicellular organs is dependent on coupling the generation of cellular heterogeneity with structural organization. My thesis exploits the retina of the genetically tractable Drosophila melanogaster to address how organs develop from naïve tissues. During retinal development a wave of apical constriction, known as the morphogenetic furrow (MF), advances across the eye-disc epithelium as the leading front of photoreceptor (PR) differentiation. My thesis addresses three aspects of organogenesis from the perspective of neurogenesis: (1) ‘Who’ will develop as a PR? (2) ‘Where’ will neurogenesis initiate? (3) ‘When’ will neurogenesis occur? ‘Who?’ Neurogenesis begins with proneural factor expression, which assigns neural competence to naïve cells; this is coupled to MF progression in the retina. The Notch pathway refines neural competence to single evenly spaced PRs, in a process termed lateral inhibition. I identified nemo (nmo) as a target of proneural factors and show that Nmo promotes Notch-mediated lateral inhibition, thus contributing to the selection of single PRs and other neural cell types. ‘Where?’ A prerequisite to the initial onset of PR neurogenesis is regional-fate competence. Antennal/head fate selectors compete with and mutually antagonize eye-fate selectors to subdivide the tissue. Overlaid onto this are signalling pathways, which cooperate to promote MF initiation and neurogenesis. I find that compromising one of these pathways, the Ras/MEK/MAPK cascade, affects where neurogenesis initiates by regulating both regional-fate specification and signal transduction. This is achieved via factors that control extracellular ligand diffusion. ‘When?’ Although signalling pathways regulate gene expression to promote MF progression and neurogenesis, how this translates into epithelial morphogenesis is not known. I find that the MF’s pace is sensitive to integrin adhesion receptor levels, and that integrins are genetically downstream of the signalling events driving MF progression. Integrins within the MF stabilize microtubules to promote apical constriction and MF progression, thus genetically linking signalling events with cytoskeletal remodeling events necessary for morphogenesis and neurogenesis. My thesis supports the notion that cellular diversity follows from the interplay between signal transduction and cell competence, and that tissue structure is a consequence of signal transduction-regulated cell adhesion and cytoskeletal remodeling.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Esther Verheyen
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.