Biochemistry, Biophysics, and Structural Biology Commons^™

Characterization Of Ranbpm Subcellular Localization And Function In Hdac6 Regulation, Louisa M. Salemi

Electronic Thesis and Dissertation Repository

RanBPM has been shown to interact with numerous proteins implicating it in a variety of cellular processes including apoptosis, transcription regulation, cell migration, adhesion and morphology and has been shown to have tumour suppressive functions. RanPBM has been hypothesized to be a scaffolding protein and is part of a large protein complex termed the CTLH complex. Although the homologous complex in yeast has been demonstrated to have E3 ubiquitin ligase activity, whether the evolutionarily conserved human complex retains this activity remains to be determined. In this work we aim to characterize the regions of RanBPM that regulate its subcellular localization …

Using Mutual Information To Reprogram Dna Specificity Of Laglidadg Endonucleases, Marcon Laforet

Electronic Thesis and Dissertation Repository

A systematic approach to reprogram protein-DNA interactions has yet to be discovered. This study investigates the ability of co-variation analyses to identify potential protein-DNA contacts that regulate specificity. Here, 27 LAGLIDADG Homing Endonucleases (LHEs) and their 22-basepair DNA targets were collated into a Multiple Sequence Alignment (MSA) that was subjected to pairwise co-variation calculations. Using the LHE I-OnuI as a reference, an amino acid-DNA pair, lysine (K) 231 and adenine +3, generated the highest score. To test if the K231/A3 score was biologically relevant we tested protein mutants for altered nuclease specificity at +3 DNA point mutants. Randomizing the 231 …

Insights Into Chibby's Structural Elements And Their Interplay In Wnt Signaling Protein-Protein Interactions, Ryan C Killoran

Electronic Thesis and Dissertation Repository

The Wnt/b-catenin signaling pathway is critical to embryonic development and adult tissue homeostasis. Mutations to Wnt signaling components can cause dysregulation of the pathway, leading to various human diseases such as cancer. The partially disordered protein Chibby (Cby) is a conserved nuclear protein that acts as an antagonist in the Wnt/b-catenin signaling pathway. Cby’s antagonism is accomplished via two mechanisms. First, by competing with the Tcf/Lef family of transcription factors, Cby abrogates the b-catenin-mediated transcription of Wnt signaling genes. Moreover, upon phosphorylation on serine 20 by the kinase Akt, Cby forms a complex with the protein 14-3-3 to facilitate the …

Modulating Parkin E3 Ubiquitin Ligase Activity Using Phospho-Ubiquitin Variants, Susanna George

Electronic Thesis and Dissertation Repository

Parkin is a Parkinson’s disease-linked E3 ubiquitin (Ub) ligase that promotes mitophagy by ubiquitination of mitochondrial outer membrane proteins. Phosphorylation of Ub at Ser65 by the PTEN-induced putative kinase 1 activates parkin. The role of other Ub phosphorylation sites and the associated kinases remain unknown. We optimized genetic code expansion to produce pure site-specfically phosphorylated Ub (pUb) variants (pUb^S7, pUb^S12, pUb^S20, pUb^S65) and investigated their activity in a key neurodegenerative pathway. Purification of pUb^S7 revealed a +3 frameshifted protein (Ub ∆7) that was successfully purified away from the pUb. Parkin was …

The Mechanism Of Activation Of Potassium Dependent And Potassium Independent Asparaginases From Common Bean (Phaseolus Vulgaris), Ebenezer Oluwafemi Ajewole

Electronic Thesis and Dissertation Repository

L-asparaginases play an important role in nitrogen mobilization in plants. This study investigated the biochemical and biophysical properties of potassium dependent (PvAspG1) and potassium independent (PvAspG-T2) L-asparaginases from P. vulgaris. Previous studies revealed that PvAspG1 requires potassium for catalytic activation and crystal structure analysis suggested that Ser-118 in the activation loop plays a critical role in alkali metal coordination. This amino acid residue is replaced by an isoleucine in PvAspG-T2. Reciprocal mutants of the enzymes were produced and the effect of the amino acid substitution on the kinetic parameters, secondary structure conformation, allosteric effector binding and pH profile were studied. …

Structural Analysis Of Tpr Ligand Complexes Of Stip1 Implicated In Alzheimer's Disease, Andrzej Maciejewski

Electronic Thesis and Dissertation Repository

Amyloid-beta oligomers (AbOs) induce neurological dysfunction in part through the cellular prion protein (PrP^C) resulting in deregulation of Ca²⁺ homeostasis in Alzheimer’s disease (AD). Stress inducible phosphoprotein 1 (STIP1), a cochaperone of Hsp70 and Hsp90, protects neurons from AbO-induced cell death. As well, STIP1 interacts with the Ca²⁺ sensor S100A1, which is an important biomarker upregulated in AD and regulates STIP1 and other cochaperone association with Hsp70 and Hsp90. While the molecular details of STIP1-Hsp complexes are well studied, little information is available concerning alternate STIP1 binding partners. Here, we investigated the structural details of STIP1 …

Domains Of Stip1 Responsible For Regulating Prpc-Dependent Amyloid-Β Oligomer Toxicity., Andrzej Maciejewski, Valeriy G Ostapchenko, Flavio H Beraldo, Vania F Prado, Marco A M Prado, Wing-Yiu Choy

Biochemistry Publications

Soluble oligomers of amyloid-beta peptide (AβO) transmit neurotoxic signals through the cellular prion protein (PrP(C)) in Alzheimer's disease (AD). Secreted stress-inducible phosphoprotein 1 (STIP1), an Hsp70 and Hsp90 cochaperone, inhibits AβO binding to PrP(C) and protects neurons from AβO-induced cell death. Here, we investigated the molecular interactions between AβO and STIP1 binding to PrP(C) and their effect on neuronal cell death. We showed that residues located in a short region of PrP (90-110) mediate AβO binding and we narrowed the major interaction in this site to amino acids 91-100. In contrast, multiple binding sites on STIP1 (DP1, TPR1 and TPR2A) …

Characterization Of Numb As A Regulator Of Anaplastic Lymphoma Kinase, Ran Wei

Electronic Thesis and Dissertation Repository

Cellular events rely on protein-protein interactions that are often mediated by modular domains which recognize particular sequence motifs in binding partners. The NUMB protein is the first described cell fate determinant and multifaceted adaptor that is involved in a wide variety of cellular events. NUMB mainly mediates protein interactions via its modular PTB domain. Here we present a systematic investigation of the NUMB-PTB interactome by employing an integrative strategy combining both protein and peptide arrays. We profiled NUMB-PTB binding specificity and interacting proteins genome-wide. The receptor tyrosine kinases (RTKs) are found highly enriched in the interactome, raising the possibility that …

Structure And Function Relationships Between Atpase Family, Aaa Domain Containing Protein 5, Proliferating Cell Nuclear Antigen, And Usp1-Associated Factor 1, Tam T. Bui

Electronic Thesis and Dissertation Repository

The genome is constantly damaged by intracellular and extracellular factors. At sites of DNA damage, replication forks are stalled, leading monoubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination of PCNA promote the switch from regular high-fidelity polymerases to Y-family polymerases for bypassing damaged DNA. Prolonged replication by these polymerases may lead to increased mutagenesis, so tight regulation of this process is required. ATAD5 recruits a deubiquitinase complex consisting of ubiquitin-specific protease 1 (USP1) and USP1-associated factor 1 (UAF1) to control PCNA monoubiquitination. The mechanism by which ATAD5 and PCNA interact has been previously unexplored. We show through biochemical and structural …

Parkin Misfolding, Dysfunction, And Degradation In Parkinson's Disease, Alexander S. Mccarton

Electronic Thesis and Dissertation Repository

Mutations in the gene encoding parkin (PARK2) result in familial early onset forms of Parkinson’s disease (PD) based on the loss of parkin’s E3 ubiquitin ligase function. Protein misfolding is a common molecular feature of most neurodegenerative diseases, including PD. To test whether parkin misfolding also plays a role in the more common spontaneous PD, we established and functionally characterized a parkin yeast model. We found that oxidative and protein folding stress, parkin point mutations and truncations, and parkin’s interaction with the PD-associated kinase PINK1 profoundly alter parkin’s subcellular localization and toxicity. Notably, these conditions also induce parkin fragmentation, degradation, …

Interaction Of Seca With Unfolded Polypeptides, Aliakbar Khalili Yazdi

Electronic Thesis and Dissertation Repository

The evolutionarily well-conserved SecA is essential for bacterial post-translational translocation. SecA uses the energy of ATP to drive preproteins through the membrane pore. The functional oligomeric state of SecA and the molecular basis for recognition of unfolded polypeptides by SecA are major unresolved questions that must be addressed to understand preprotein targeting and the molecular mechanics of SecA-mediated translocation. This thesis will address three aspects of these questions. First, the role of unstructured termini in the oligomerization and function of various SecA constructs was elucidated. By re-examining the tetramerization of a truncated SecA construct (SecA-N68), it was shown that the …

The Role Of Sirtuin 6 In Maintaining Vascular Integrity, Sharon Z. Leung

Electronic Thesis and Dissertation Repository

Oxidative stress is an underlying cause for vascular pathologies including inflammation, hypertension, and atherosclerosis. Sirtuins (SIRTs) are a family of NAD+ dependent deacetylases with pronounced roles in cellular metabolism and aging. SIRT6 is expressed in vascular smooth muscle cells (SMCs) and may offer protection from oxidative stress-induced damage. To study the role of SIRT6 in SMCs, we created a novel strain of SMC-specific SIRT6-deficient (SIRT6KO) mice with Cre-lox technology. Because no defects were observed in the aortas of SIRT6KO mice, they were then infused with angiotensin II (Ang II) to induce oxidative stress. Compared with vehicle controls, SIRT6KO mice developed …

A Unified Analytic Framework For Prioritization Of Non-Coding Variants Of Uncertain Significance In Heritable Breast And Ovarian Cancer, Eliseos J. Mucaki, Natasha G. Caminsky, Ami M. Perri, Ruipeng Lu, Alain Laederach, Matthew Halvorsen, Joan H. M. Knoll, Peter K. Rogan

Biochemistry Publications

Background

Sequencing of both healthy and disease singletons yields many novel and low frequency variants of uncertain significance (VUS). Complete gene and genome sequencing by next generation sequencing (NGS) significantly increases the number of VUS detected. While prior studies have emphasized protein coding variants, non-coding sequence variants have also been proven to significantly contribute to high penetrance disorders, such as hereditary breast and ovarian cancer (HBOC). We present a strategy for analyzing different functional classes of non-coding variants based on information theory (IT) and prioritizing patients with large intragenic deletions.

Methods

We captured and enriched for coding and non-coding variants …

Cost-Effectiveness Of Using A Gene Expression Profiling Test To Aid In Identifying The Primary Tumour In Patients With Cancer Of Unknown Primary., M B Hannouf, E Winquist, S M Mahmud, M Brackstone, S Sarma, G Rodrigues, P Rogan, J S Hoch, G S Zaric

Biochemistry Publications

We aimed to investigate the cost-effectiveness of a 2000-gene-expression profiling (GEP) test to help identify the primary tumor site when clinicopathological diagnostic evaluation was inconclusive in patients with cancer of unknown primary (CUP). We built a decision-analytic-model to project the lifetime clinical and economic consequences of different clinical management strategies for CUP. The model was parameterized using follow-up data from the Manitoba Cancer Registry, cost data from Manitoba Health administrative databases and secondary sources. The 2000-GEP-based strategy compared to current clinical practice resulted in an incremental cost-effectiveness ratio (ICER) of $44,151 per quality-adjusted life years (QALY) gained. The total annual-budget …

Automated Discrimination Of Dicentric And Monocentric Chromosomes By Machine Learning-Based Image Processing., Yanxin Li, Joan H Knoll, Ruth C Wilkins, Farrah N Flegal, Peter K Rogan

Biochemistry Publications

Dose from radiation exposure can be estimated from dicentric chromosome (DC) frequencies in metaphase cells of peripheral blood lymphocytes. We automated DC detection by extracting features in Giemsa-stained metaphase chromosome images and classifying objects by machine learning (ML). DC detection involves (i) intensity thresholded segmentation of metaphase objects, (ii) chromosome separation by watershed transformation and elimination of inseparable chromosome clusters, fragments and staining debris using a morphological decision tree filter, (iii) determination of chromosome width and centreline, (iv) derivation of centromere candidates, and (v) distinction of DCs from monocentric chromosomes (MC) by ML. Centromere candidates are inferred from 14 image …

Prioritizing Variants In Complete Hereditary Breast And Ovarian Cancer (Hboc) Genes In Patients Lacking Known Brca Mutations., Natasha G Caminsky, Eliseos J Mucaki, Ami M Perri, Ruipeng Lu, Joan H M Knoll, Peter K Rogan

Biochemistry Publications

BRCA1 and BRCA2 testing for Hereditary breast and ovarian cancer (HBOC) does not identify all pathogenic variants. Sequencing of 20 complete genes in HBOC patients with uninformative test results (N = 287), including non-coding and flanking sequences of ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD51B, STK11, TP53, and XRCC2, identified 38,372 unique variants. We apply information theory (IT) to predict and prioritize non-coding variants of uncertain significance (VUS) in regulatory, coding, and intronic regions based on changes in binding sites in these genes. Besides mRNA splicing, IT provides a common …

Examining The Role Of Atrx In Astrocytes, Haley Mcconkey

Electronic Thesis and Dissertation Repository

Astrocytes perform many homeostatic roles in the brain while supplying metabolites to neurons and mediating synaptic transmission. The current study explored a possible role of the Atrx gene in astrocytes. Hypomorphic mutations in this gene cause the ATR-X intellectual disability syndrome. Deletion of Atrx in the forebrain leads to an apparent increase in reactive astrocytes, potentially caused by the high level of neuroprogenitor cell death. To avoid such non cell-autonomous effects on astrocytes, we generated mice with inducible conditional inactivation of Atrx in astrocytes. Preliminary analysis two weeks following induction of Atrx gene deletion revealed variably lower expression of Connexin …

Genomic Signatures For Paclitaxel And Gemcitabine Resistance In Breast Cancer Derived By Machine Learning., Stephanie N Dorman, Katherina Baranova, Joan H M Knoll, Brad L Urquhart, Gabriella Mariani, Maria Luisa Carcangiu, Peter K Rogan

Biochemistry Publications

Increasingly, the effectiveness of adjuvant chemotherapy agents for breast cancer has been related to changes in the genomic profile of tumors. We investigated correspondence between growth inhibitory concentrations of paclitaxel and gemcitabine (GI50) and gene copy number, mutation, and expression first in breast cancer cell lines and then in patients. Genes encoding direct targets of these drugs, metabolizing enzymes, transporters, and those previously associated with chemoresistance to paclitaxel (n = 31 genes) or gemcitabine (n = 18) were analyzed. A multi-factorial, principal component analysis (MFA) indicated expression was the strongest indicator of sensitivity for paclitaxel, and copy number and expression …