Browsing by Subject "Mutation"
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Item The CFTR Folding Pathway: Implications for the Identification and Development of CF Therapeutics(2012-07-20) Mendoza, Juan Luis; Thomas, Philip J.The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is a member of the ABC transporter superfamily, important for Cl- conductance at the apical cell membrane. Loss-of-function of CFTR leads to Cystic Fibrosis (CF), a fatal genetic disease affecting 70,000 people worldwide. There are hundreds of CF causing mutations with the most common being ΔF508, present in at least one allele in 90% of CF patients. CFTR, comprising of 1480 amino acids, folds into five domains important for forming the channel through the membrane, and the regulation of channel function. F508 is located in Nucleotide Binding Domain 1 (NBD1) and is predicted to be at the interface with Intracellular Loop 4 (ICL4) of Transmembrane Domain 2 (TMD2). Studies of the isolated NBD1 demonstrate that the ΔF508 mutation impacts the folding pathway and stability of the domain. Misfolding of NBD1 contributes to the trafficking defect of the intact protein and subsequent loss-of-function. Conversely, second-site suppressor mutations, which more than compensate for defects of the mutant NBD1 domain, only partially rescue CFTR trafficking, suggesting that the deletion also affects other steps along the folding pathway. The aim of this work was to identify positions in CFTR critical for defining the folding pathway. We used a computational approach and two in vitro folding assays to monitor folding of the isolated NBD1 domain and trafficking of full-length CFTR. These data establish a correlation between the folding of the isolated NBD1 domain and maturation of full-length CFTR. Further, NBD1 second-site suppressor mutations in the ΔF508, F508K (NBD1/ICL4 interface disrupting mutation), and R1070W (ΔF508 NBD1/ICL4 interface stabilizing mutation) backgrounds suggest that ΔF508 CFTR is defective in two steps of CFTR biogenesis: 1) stability and efficiency of folding of the NBD1 domain, and 2) NBD1/ICL4 docking. We demonstrate that efficient rescue of ΔF508 CFTR requires correction the two distinct defects. This work has implications for the discovery and development of CF therapeutics by providing a framework for understanding the observed ceiling in the efficacy of either suppressor mutations or corrector compounds, which likely correct a single defect.Item Characterization of the T122L Mutation in p53 and Its Protein Product in XPC Mutant Mice(2003-04-01) Nahari, Dorit; Friedberg, Errol C.Xeroderma Pigmentosum (XP) is a rare genetic disorder characterized by extreme sensitivity to sunlight, and a profound predisposition to skin cancer due to defects in nucleotide excision repair (NER) of DNA. XP patients can be divided into seven complementation groups (A-G) with corresponding genes Xpa thru Xpg. I have studied a novel UV-induced hot spot in codon 122 of the p53 gene in mice deficient in XPC protein and heterozygous for the p53 gene. The original Threonine residue is mutated to Leucine as a result of an AC->TT change. The main goal of this work was to characterize the T122L mutation and its protein product, and to elucidate the mechanism(s) that affect its appearance in XPC deficient skin. I have shown that the T122L mutation is rare in other NER deficient mouse models, suggesting that the XPC protein is required specifically to repair the unidentified damage in this codon. In addition, I have shown that the T122L mutant protein is not a loss of function mutant, but it retains some wild type protein functions including ransactivation of p53 regulated genes and promotion of cell cycle arrest in response to UV-induced DNA damage. The altered transactivation properties of the mutant protein might support clonal expansion, giving cells that express it a growth advantage. In order to determine whether the p53 mutant protein function contributes significantly to the elevated mutation frequency observed in XPC deficient mice I have used a p53 knockout mouse model that retains the genomic region containing codon 122 without expressing p53 protein. I show that the mutation is observed in low frequencies in mice that do not express protein, suggesting that repair deficiency is the key factor for the appearance of the T122L mutation. However, once the mutation is formed its frequency is significantly increased as a result of the mutant protein function. The specific requirement for XPC protein and the location of the damage at a nondipyrimidine nucleotide site hints at an additional role of XPC in a repair pathway different from NER.Item Colon Cancer Initiation and Progression(2012-07-20) Eskiocak, Ugur; Shay, Jerry W.Landmark cancer genome resequencing efforts are leading to the identification of mutated genes in many types of cancer.The extreme diversity of mutations being detected presents significant challenges to subdivide causal from coincidental mutations in order to elucidate how disrupted regulatory networks drive cancer processes. Given that a common early perturbation in solid tumor initiation is bypass of matrix-dependent proliferation restraints we sought to functionally interrogate candidate colorectal cancer genes (CAN-genes) to identify driver tumor-suppressors. We have developed an isogenic human colonic epithelial cell (HCEC) model to identify suppressors of anchorage-independent growth by conducting a soft agar based shRNA screen within the cohort of CAN-genes. Remarkably, depletion of 65 of the 151 CAN-genes tested collaborated with ectopic expression of K-RASV12 and/or TP53 knockdown to promote anchorage-independent proliferation of HCECs. In contrast only 5 out of 362 random shRNAs (1.4%) enhanced soft agar growth. We have identified additional members of an extensive gene network specifying matrix-dependent proliferation, by constructing an interaction map of these confirmed progression suppressors with the ~700 mutated genes that were excluded from CAN-genes, and experimentally verifying soft-agar growth enhancement in response to depletion of a subset of these genes. Collectively, this study revealed a profound diversity of nodes within a fundamental tumor suppressor network that are susceptible to perturbation leading to enhanced cell-autonomous anchorage-independent proliferative fitness. Tumor suppressor network fragility as a paradigm within this and other regulatory systems perturbed in cancer, could in large part, account for the heterogeneity of somatic mutations detected in tumors.Item Genetic Mutations: A STARS Scientific Suitcase for 9th Grade High School Science Educators(2011-12-13) Lankes, Richard Thomas; Krumwiede, Kimberly HoggattThe purpose of this project was to design and produce a lightweight scientific suitcase teaching tool that ninth grade high school science educators may implement into their curriculum when teaching students the concepts about genetic mutations. Contained within the suitcase are an animation, card game, hands-on models, and display posters, along with a teacher’s instruction manual. This scientific suitcase was created in an attempt to fill in the apparent lack of information over genetic mutations that is present in current Texas high school textbooks and resources. It may assist students in better preparing for standardized testing by giving their educators an all-in-one module that can give the classroom extensive information on genetic mutations, all in one easy to carry suitcase. The suitcase components have been evaluated for their effectiveness and appeal by current educators, from 8th grade to college level, who specialize in multiple fields of science. The scientific suitcase’s impact on students’ performance and comprehension will be tested by STARS and DISD once it has been fully integrated into the science classroom curriculum The purpose of this project was to design and produce a lightweight scientific suitcase teaching tool that ninth grade high school science educators may implement into their curriculum when teaching students the concepts about genetic mutations. Contained within the suitcase are an animation, card game, hands-on models, and display posters, along with a teacher’s instruction manual. This scientific suitcase was created in an attempt to fill in the apparent lack of information over genetic mutations that is present in current Texas high school textbooks and resources. It may assist students in better preparing for standardized testing by giving their educators an all-in-one module that can give the classroom extensive information on genetic mutations, all in one easy to carry suitcase. The suitcase components have been evaluated for their effectiveness and appeal by current educators, from 8th grade to college level, who specialize in multiple fields of science. The scientific suitcase’s impact on students’ performance and comprehension will be tested by STARS and DISD once it has been fully integrated into the science classroom curriculumItem Hepatitis B virus infections: New mutations, new drugs(2003-04-24) Ocama, Ponsiano; Lee, William M.Item Molecular Characterization of Novel FOXN1 Mutations Causal to Thymic Aplasias in Human Patients(2018-01-23) Huynh, Larry; Du, Qiumei; Khan, Shaheen; Molina, Erika; Padron, Grace T.; Markert, M. Louise; de la Morena, Maria Teresa; van Oers, NicolaiThe Forkhead Box N1 (FOXN1) transcription factor plays a crucial role in thymic epithelial cell development. Humans and mice harboring FOXN1 mutations have a profound T-cell deficiency caused by their thymic aplasia. They also present with alopecia and nail dystrophy. Recently, two patients were identified with T-cell immunodeficiency. Both patients have normal hair and nailbed development. Genetic workup revealed that each patient carried distinct compound heterozygous mutations in FOXN1 not previously reported. Molecular characterization of these FOXN1 mutations will provide new insight into how this transcription factor functions in thymus development. To characterize these mutations, CRISPR/Cas9 technologies were used to create similar compound heterozygous mutations in mouse models. The mice are currently being intercrossed to determine the impact of these novel FOXN1 mutations on thymus development. To determine how these mutations impact FOXN1 function, we undertook transcriptional reporter assays. Preliminary results suggest only one of these mutations led to loss of transcriptional activity. Western blot analysis indicated that this mutation led to a truncation of the protein. Further experiments including co-immunoprecipitation assays, transcriptome analyses, and functional studies will reveal how these compound heterozygous mutations impact the functions of FOXN1. Findings from this study may lay the foundation for novel therapeutic strategies at restoring thymopoiesis in a number of distinct clinical settings. These can include patients undergoing radiation treatment, chemotherapy, and in any other conditions that can lead to a thymic aplasia.Item Mutant Secretagogin, a Potential Cause of Ulcerative Colitis, Exhibits Reduced Affinity for SNARE Complex Protein SNAP-25(2016-01-19) Norris, Nicholas; Sifuentes-Dominguez, Luis; Burstein, EzraINTRODUCTION: Inflammatory Bowel Diseases (IBD) encompass a set of poorly understood multifactorial inflammatory disorders including Chrohn's disease (CD) and Ulcerative Colitis (UC). To date, around 200 genetic variants associated with increased risk of IBD have been identified through genome-wide association studies. Recently, our lab identified three siblings with UC from consanguineous parents who were all homozygous for a rare hypomorphic variant in the gene SCGN which encodes the calcium sensing protein secretagogin. Within the GI tract, secretagogin is localized specifically to enteroendocrine cells (EECs) suggesting an unforeseen role of EECs in IBD pathogenesis. This disease associated variant results in an amino acid substitution (R77H) in the Ca2+ binding region of secretagogin, which has been shown to interact with SNARE proteins in a Ca2+ dependent manner to mediate membrane fusion and exocytosis. Therefore, we examined whether this SCGN mutation impairs its Ca2+ dependent binding to the SNARE complex component SNAP-25 in vitro. METHODS: A GST pull-down assay was performed using recombinant GST fusion proteins of human secretagogin (WT or R77H) immobilized to glutathione-agarose beads. These beads were used to affinity purify SNAP-25 from whole cell lysates of STC-1 cells, a mouse enteroendocrine tumor cell line. Because SNAP-25 interactions with secretagogin are Ca2+ dependent, whole cell lysates were prepared in either Ca2+ rich or Ca2+ free conditions. The beads were washed and bound proteins were detected by SDS-PAGE and western blot analysis using appropriate antibodies. RESULTS: Under Ca2+ conditions, SNAP-25 was successfully bound to secretagogin, but the amount of SNAP-25 bound to mutant R77H secretagogin was significantly less. As expected, SNAP-25 did not bind secretagogin under Ca2+ free conditions. CONCLUSIONS: We confirm secretagogin interacts with SNAP-25 in a Ca2+ dependent fashion and the R77H mutation, located in the Ca2+ binding pocket of secretagogin, disrupts this interaction representing a hypomorphic mutation. This finding is consistent with the recessive nature of this mutation and strengthens its physiological relevance. Furthermore, it suggests that impaired exocytosis and hormone release from EECs contributes to the disease pathogenesis. Further study to elucidate which (if any) EEC secretory products play key roles in IBD prevention is needed. Understanding potentially aberrant paracrine mechanisms underlying IBD could open the door to novel treatments like hormone replacement therapy.Item Mutations in G proteins and G protein-coupled receptors in human disease: implications for diagnosis and treatment(2020-10-09) Spiegel, Allen M.Item Regulation of the cGAS-STING Pathway in Health and Disease(2018-11-27) Pokatayev, Vladislav Andreyevich; van Oers, Nicolai S. C.; Conrad, Nicholas; Chen, Zhijian J.; Yan, NanThe innate immune system senses non-self or altered-self molecular structures through pattern recognition receptors in order to eliminate pathogens or damaged cells, and restore an organism to its basal physiology. Nearly all nucleated cell types can sense intracellular viral nucleic acids. These sensors detect either viral RNA through RIG-I like receptors or DNA through the cGAS-STING signaling pathway. Antiviral immune pathways are vital for resolution of viral infections; however, their dysregulation may give rise to various immune-mediated diseases. The neuro-inflammatory autoimmune disease Aicardi-Goutières Syndrome (AGS) develops from mutations in genes encoding several nucleic acid processing proteins, including RNase H2. Defective RNase H2 may induce accumulation of self-nucleic acid species which trigger chronic inflammation leading to AGS pathology. We created a knock-in mouse model with an RNase H2 AGS mutation in a highly conserved residue of the catalytic subunit, Rnaseh2aG37S/G37S (G37S), the most severe Rnaseh2a mutation categorized as it abolishes nuclease activity to less than 10% of WT RNase H2, to understand disease pathology. Importantly, I found that the G37S mutation induces a cellular anti-viral state, and an increased expression of interferon-stimulated genes dependent on the cGAS-STING signaling pathway. G37S homozygotes are perinatal lethal, and ablation of STING in G37S mice results in partial rescue of the perinatal lethality and complete rescue of the immune phenotype. This study motivates inhibitors of the cGAS-STING pathway in the goal of resolving Rnaseh2a-mediated AGS. As my previous work implicates STING in the development of AGS, I performed a genetic screen to identify novel regulators of this protein. I discovered that TOLLIP, a protein previously identified as a regulator of extracellular Toll-like receptor pathways, can function as a positive regulator of the cGAS-STING pathway. TOLLIP antagonizes STING protein degradation through a regulatory pathway controlled by the protein IRE1α. In Tollip-/- cells, IRE1α is activated and induces lysomal-mediated degradation of STING. Chronic activation of this degradative pathway blunts the cellular response to cGAS or STING agonists. These findings have implications in vivo, as deleting Tollip in a mouse model for AGS, the Trex1-/- mouse, can rescue symptoms of the disease. These findings have clinical importance, as novel therapeutics against TOLLIP can be developed to treat auto-inflammation caused by dysregulation of the cGAS-STING signaling pathway.Item The Role of SHANK3 at the Synapse and Its Implications in Autism-Associated Behaviors and Synaptic Transmission(2015-04-10) Kouser, Mehreen; Rothenfluh, Adrian; Huber, Kimberly M.; Bibb, James A.; Powell, Craig M.Autism is a neurodevelopmental disorder characterized by an increase in repetitive behaviors and impairments in social interaction and communication. Since its discovery, a multitude of studies have linked SHANK3 to autism. Moreover, deletion of SHANK3 has been shown to cause Phelan McDermid Syndrome (22q13 Deletion Syndrome) by several human studies. Shank3 is a multi domain post-synaptic scaffolding proteins that is found in excitatory synapses and plays a critical role in forming the post-synaptic density by connecting the necessary machinery together. In this study, I have characterized a homozygous Shank3 mutation in mice that deletes exon 21(Shank3ΔC) including the Homer binding domain. In the homozygous state, deletion of exon 21 results in loss of the major, naturally occurring Shank3 protein bands. Shank3ΔC/ΔC mice exhibit an increased localization of mGluR5 to the synapses in the hippocampus, a decrease in NMDA/AMPA excitatory postsynaptic current ratio in area CA1 of hippocampus, reduced long-term potentiation in area CA1, and deficits in hippocampus-dependent spatial learning and memory. In addition, these mice also exhibit motor-coordination deficits, hypersensitivity to heat, novelty avoidance, altered locomotor response to novelty, and minimal social abnormalities. I also report on a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3G) which causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse like the Shank3ΔC/ΔC mice. Shank3G/G mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, and altered response to novelty. Shank3G/G mice also exhibit impaired hippocampal excitatory transmission and plasticity. Finally, Shank3G/G mice were designed to be genetically rescued to wild-type at various times during development. In this study, I also report on the biochemical and behavioral results of the genetic rescue in Shank3G/G mice after the completion of neurodevelopment. I was able to achieve a biochemical rescue in the Shank3G/G mice. Interestingly, not all the behavioral impairments observed in Shank3G/G mice were replicated in the Reversible-Shank3G/G mutation mice making the interpretation of the data more challenging which is discussed in detail in this thesis.Item Searching for Genes of Host Defense(2013-01-22) SoRella, Jeffrey A.; Shi, He-Xin; Wang, Ying; Beutler, Bruce A.Through random mutation of the mouse genome and phenotypic screening of the mutated mice, genes can be identified that are associated with dysfunction in the innate immune system. The strategy proposed works under the knowledge that many genes are involved in the immune system and that random mutation could lead to a change in their genetic code. This mutation can present as a phenotypically abnormal immune system. Once a phenotype is identified, the genome can be analyzed in an attempt to trace the mutated gene responsible for the weakened immune system. One of the elegant aspects of this genetic method is that it does not rely on a hypothesis about how the immune response works. This leads to an unbiased approach where interpretation errors are rarely made. A forward genetic approach is used to create abnormal phenotypes of the innate immune system and then determine the genetic cause. The normal mutation rate is accelerated by the widely used germline mutagen N-ethyl-N-nitrosourea (ENU) to produce an average of 3,000 single nucleotide changes per host leading to an average of 60 coding changes. To produce homozygotes, males of the G1 generation are bred with normal mice of the same strain to yield the G2 generation. Recessive mutations can be found in the G3 generation by a backcross of G2 females with the G1 father. Screening 6 G3 progeny should capture 50% of the mutations in the homozygous form. Phenotypic screening was performed on peritoneal macrophages ex vivo by stimulation with the following toll-like receptor agonists: lipopolysaccharide (TLR4), double stranded RNA (TLR3), triacylated lipoprotein (TLR 1/2), diacylated lipoprotein (TLR 2/6), resiquimod (TLR 7), and unmethylated DNA (TLR 9). The inflammasome pathway was probed by lipopolysaccharide priming followed by stimulation with either nigericin (K+ efflux) or ATP. The secreted TNF-alpha (TLR screen) and IL-1beta (inflammasome screen) were measured by ELISA to determine phenovariance. This research can lead to a deeper understanding of how we combat infection. The study can lead to the development of mutations involved in both the innate and adaptive immune system so autoimmune diseases can also be studied. A long term goal is to identify genes that would render an individual resistant to infection and to study the interaction of these genes.Item [Southwestern News](2003-07-17) Shields, AmyItem [Southwestern News](2003-07-06) Siem, Staishy BostickItem [Southwestern News](2001-04-27) Shields, AmyItem [Southwestern News](2003-07-14) Maier, ScottItem [Southwestern News](2004-08-30) Siegfried, AmandaItem [Southwestern News](2005-07-17) Siegfried, Amanda; Watson, JohnItem [Southwestern News](1998-11-24) Steeves, Susan A.Item [Southwestern News](1998-12-11) Steeves, Susan A.Item Transmembrane Protein Folding: Effects of Disease-Causing Mutations on CFTR Folding and Assembly(2006-05-16) Thibodeau, Patrick Harlan; Thomas, Philip J.The biosyntheses of multi-domain membrane proteins are complex processes which involve the translation, folding, and assembly of domains to reach the native state. The nascent chain of a membrane protein must interact with multiple solvent environments, ribosome and chaperone components, and processing and trafficking machinery, and each of these steps are at least partially determined by the protein sequence and structure. Alterations to protein sequences often perturb these processes by impacting any of a number of structural states of the protein, and while some mutations impact the native state structures of proteins directly, others impact the folding process and have little direct effect on the native state structure. A growing number of mutations have been shown to impact these folding processes in the cystic fibrosis transmembrane conductance regulator (CFTR), a multi-domain transmembrane protein associated with cystic fibrosis. Two such mutations are detailed in this work: F508del and P205S. The most common CF-causing mutation, F508del, is the deletion of a single phenylalanine residue in a cytosolic domain of CFTR and results in a protein which fails to fold at physiological temperature, is retained in the ER and is degraded by the proteasome. The resulting loss of protein is the underlying basis for cystic fibrosis. The loss of the backbone at this position induces the misfolding of the domain, while changes in sidechain character impact subsequent domain-domain assembly. The rescue of full-length F508del CFTR by second-site suppressors correlates with the rescue of the folding of the soluble domain, further suggesting the direct role of Phe508 in domain folding. The mutation of equivalent residues in homologous proteins results in similar phenotypes, suggesting an evolutionary conservation of function for this position. The Pro205 residue, in the first transmembrane domain, has been shown to facilitate proper folding by disfavoring alternate, non-native protein conformations. A computational study of proline residues in transmembrane helices suggests that this mechanism is also conserved evolutionarily. With these data, a hierarchical model for CFTR folding is presented and mechanisms by which these mutations specifically impact the stepwise folding and assembly of CFTR are suggested.