Browsing by Department "Institute for Cellular and Molecular Biology"

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Activity-dependent regulation of ion channel gene expression: a homeostatic hypothesis for drug tolerance
(2006) Ghezzi, Alfredo; Atkinson, Nigel (Nigel S.)
Conservation of the balance between excitation and inhibition of neural activity is critically important for the proper function of the nervous system. Upon alterations in excitability, the nervous system may thus trigger mechanisms that attempt to restore homeostasis. Many alcohols, anesthetics, and other abused volatile solvents such as ethanol, benzyl alcohol, toluene, trichloroethylene, and chloroform alter neural excitability and trigger homeostatic adaptations that through the modulation of gene expression and signaling between nerve cells act to counteract these alterations. Many of these adaptations may account for the development of tolerance, dependence and addiction to these drugs. Here, I demonstrate that in Drosophila, tolerance to the sedative effects of alcohols and anesthetics, is mediated by an increase in expression of the Ca2+ activated K+ channel gene, slowpoke. A mutation that eliminates slowpoke expression prevents tolerance, while expression from an inducible slowpoke transgene mimics tolerance in naive animals. Furthermore, the behavioral and molecular response to volatile solvents can be separated into an initial phase of hyperkinesis that causes a drop in slowpoke gene expression and makes animals more sensitive to subsequent sedation by these drugs, and a sedative phase that stimulates slowpoke gene expression and induces tolerance. This demonstrates that the changes in expression level of slowpoke act as a modulator of drug sensitivity. Because of it's central role as a regulator of electrical activity in nerve terminals this channel gene is a likely contributor to the homeostatic mechanism that resists untoward changes in net cellular excitability and mediates tolerance to sedation. If hyperexcitability is induced, the proposed mechanism alters channel expression to reduce this excitability, whereas if cellular excitability is suppressed, channel gene expression changes to enhance excitability. An electrophysiological test of this hypothesis shows that increased slowpoke expression enhances the excitability of a neural pathway in a way that opposes the effects of sedative drugs. This data supports the notion that slowpoke mediated tolerance to sedation is part of a homeostatic adaptation that compensates for changes in neural activity caused by drugs and represents a step forward in the understanding of the molecular basis of drug addiction.
Anti-cancer and anti-viral aptamers
(2006) Chu, Ted Chitai; Ellington, Andrew D.
Aptamers generated by two approaches, from purified protein and whole cell surface selections, have the potential to serve as detection and therapeutic agents against prostate cancer. In addition, aptamers generated from the purified viral protein NS1 have been shown to inhibit influenza virus replication in cell culture. Anti-prostate specific membrane antigen aptamer A9 was used with high specificity for common detection methods such as flow cytometry and confocal microscopy and for novel detection methods such as proximity dependent ligation assay. Moreover, the PSMA aptamer was shown to function as a delivery vehicle for siRNA and toxin. Cell-type specific GAPDH and lamin A/C gene knockdown were demonstrated using streptavidin as a bridge to connect biotin-conjugated aptamers to biotin-conjugated siRNA. Cytotoxicity assays also revealed that aptamers were able to specifically deliver the recombinant plant toxin rGelonin specifically to LNCaP cells when conjugated with the toxin. Furthermore, aptamers were generated from whole cell surface selections of the prostate cancer cell lines LNCaP and PC3. This suggested that anti-PC3 aptamers could target cell surfaces through indirect immunostaining and images taken by confocal microscopy. Membrane binding assays, FACS, and proximate dependent ligation assays also revealed that the aptamers were able to distinguish with great sensitivity and specificity the two non-prostate cancer cell lines, A549 and H526 as well as LNCaP and PC3 cells from DU145, another common prostate cancer line. More importantly, these experiments demonstrated that aptamers could be designed to have specificities to a certain subtype of cancer cells or to the general population. In other studies, aptamers vi were also generated from purified NS1 protein encoded by the influenza virus. The calculated Kd for these aptamers was in the range of 10 nM for the full length NS1 protein. Competition and binding assays suggested that the aptamers also targeted the dsRNA binding domain of the NS1 protein. Transfecting aptamers to human lung cancer cells, A549, infected with influenza virus revealed that the aptamers were able to inhibit the replication of both the human and avian strains of influenza virus in cell cultures. These findings strongly demonstrated the broad range and effectiveness of aptamers as tools for detection, diagnostics, and therapeutics.
The application of aptamer microarraying techniques to the detection of HIV-1 reverse transcriptase and its mutant variants
(2010-08) Syrett, Heather Angel; Ellington, Andrew D.; Kitto, George B.; Willets, Katherine A.; Iyer, Vishwanath R.; Yin, Yuhui W.
The work described here details the experimental progress toward an improved means of HIV-1 diagnosis and an explanation of the experimental approaches taken to advance a previously developed HIV-1 reverse transcriptase detection assay using RNA aptamers for protein capture. After characterization of the identity and function of the aptamer samples to be used, we first set about clarifying the nature of the assay and pinning down sources of variability inherent in the original Aptamer Antibody Sandwich Assay (AASA) such that through the course of this work we might bring the assay to a point of high reproducibility. In doing so, we devised a set of criteria for data analysis and filtration and established a process to examine whether modifications to the method resulted in measurable improvement. Two new methods were tested in the hope that they might later be extended to our ultimate project goal of distinguishing binding affinity variations among HIV-1 reverse transcriptase protein and its mutant variants. Both method modifications involved the addition of a fluorescently labeled Cy5 probe to the immobilized aptamer construct. The addition of a fluorescent label to each printed aptamer allowed for detection of aptamer presence in addition to protein binding, essentially serving as a simple internal control for aptamer-protein binding. After optimizing the AASA aptamer construct and experimental procedure, the AASA was extended to a multiplexed array format. Using four groups of aptamers selected against two HIV-1 RT variants (wild-type and mutant 3) we tested the hypothesis that immobilized anti-HIV-1 aptamers might be capable of binding HIV-1 RT variants and regardless of their selective target. The experiments described here are the first example of these aptamers being used in a multiplexed array format, and the results are not only a clear exemplification of the capacity of RNA aptamers for detection in this novel, immobilized assay format, but also an indicator of the utility and flexibility of RNA aptamer functionality. The promising results described in these preliminary studies are the starting block from which several interesting aptamer-protein interaction and drug-competition studies have begun.
ArrayPlex: distributed, interactive and programmatic access to genome sequence, annotation, ontology, and analytical toolsets
(Genome Biology, 2008-11-12) Killion, Patrick J.; Iyer, Vishwanath R.
ArrayPlex is a software package that centrally provides a large number of flexible toolsets useful for functional genomics, including microarray data storage, quality assessments, data visualization, gene annotation retrieval, statistical tests, genomic sequence retrieval and motif analysis. It uses a client-server architecture based on open source components, provides graphical, command-line, and programmatic access to all needed resources, and is extensible by virtue of a documented application programming interface. ArrayPlex is available at http://sourceforge.net/projects/arrayplex/ webcite.
ATM activation by oxidative stress
(2009-08) Guo, Zhi, 1978-; Paull, Tanya T.
The Ataxia-telangiectasia mutated (ATM) protein is regarded as the major regulator of the cellular response to DNA double Strand Breaks (DSBs). In response to DSBs, ATM dimers dissociates into active monomers in a process promoted by Mre11-Rad50-Nbs1 (MRN) complex. ATM-deficient cells exhibit signs of chronic oxidative stress, suggesting that ATM plays an important role in the regulation of reactive oxygen species (ROS). I show for the first time that ATM can be activated by oxidative stress directly in the form of exposure to H₂O₂. In vitro kinase assays with purified ATM suggest that the activation by H₂O₂ is independent of DSBs and the MRN complex. In 293T cells, H₂O₂ induces ATM autophosphorylation on serine 1981. p53 and Chk2 are also phosphorylated by ATM after H₂O₂ treatment but not histone H2AX and heterochromatin protein Kap1, indicating that ATM activation by H₂O₂ in human cells is independent of DNA damage. I also show that the cysteine residue 2991 is critical for ATM activation by H₂O₂ in vitro.
A Bacteriophage Tailspike Domain Promotes Self-Cleavage of a Human Membrane-Bound Transcription Factor, the Myelin Regulatory Factor MYRF
(PLOS Biology, 2013-08-13) Li, Zhihua; Park, Yungki; Marcotte, Edward M.
Myelination of the central nervous system (CNS) is critical to vertebrate nervous systems for efficient neural signaling. CNS myelination occurs as oligodendrocytes terminally differentiate, a process regulated in part by the myelin regulatory factor, MYRF. Using bioinformatics and extensive biochemical and functional assays, we find that MYRF is generated as an integral membrane protein that must be processed to release its transcription factor domain from the membrane. In contrast to most membrane-bound transcription factors, MYRF proteolysis seems constitutive and independent of cell- and tissue-type, as we demonstrate by reconstitution in E. coli and yeast. The apparent absence of physiological cues raises the question as to how and why MYRF is processed. By using computational methods capable of recognizing extremely divergent sequence homology, we identified a MYRF protein domain distantly related to bacteriophage tailspike proteins. Although occurring in otherwise unrelated proteins, the phage domains are known to chaperone the tailspike proteins' trimerization and auto-cleavage, raising the hypothesis that the MYRF domain might contribute to a novel activation method for a membrane-bound transcription factor. We find that the MYRF domain indeed serves as an intramolecular chaperone that facilitates MYRF trimerization and proteolysis. Functional assays confirm that the chaperone domain-mediated auto-proteolysis is essential both for MYRF's transcriptional activity and its ability to promote oligodendrocyte maturation. This work thus reveals a previously unknown key step in CNS myelination. These data also reconcile conflicting observations of this protein family, different members of which have been identified as transmembrane or nuclear proteins. Finally, our data illustrate a remarkable evolutionary repurposing between bacteriophages and eukaryotes, with a chaperone domain capable of catalyzing trimerization-dependent auto-proteolysis in two entirely distinct protein and cellular contexts, in one case participating in bacteriophage tailspike maturation and in the other activating a key transcription factor for CNS myelination.
Behavioral and neuronal changes due to 13-Cis-retinoic acid treatment
(2008-05) O'Reilly, Kally Corissa; Lane, Michelle
13-Cis-retinoic acid (13-cis-RA) is a synthetic retinoid and the active ingredient in the oral acne treatment Accutane. The medical literature has suggested that the use of 13-cis-RA for acne treatment can induce depression, but because acne itself can have a negative psychosocial impact on self esteem, whether or not 13-cis-RA can cause depression remains controversial. The purpose of this work was to examine whether chronic 13-cis-RA administration could induce depression-related behaviors in mice and to determine the impact 13-cis-RA has on regions of the brain thought to be associated with mood and depression. We found that chronic treatment of adolescent male mice with 13-cis-RA induced depression-related behaviors, as assessed by immobility in the tail suspension and forced swim tests. Although depression is a multifaceted disease in which many brain regions are involved, the regions that seem particularly vulnerable to the effects of 13-cis-RA are the serotonergic and hippocampal systems. In serotonergic cells in vitro, 13-cis-RA treatment increases protein levels of the serotonergic 5-HT[subscript 1A] autoreceptor and the serotonin reuptake transporter (SERT), two inhibitory components of serotonin (5-HT) signaling. In vivo, the median and dorsal raphe nuclei contain the main 5-HT producing cells. 13-Cis-RA uncoupled the functional connectivity of dorsal raphe nuclei from the hippocampal regions as measured by interregional correlations of cytochrome oxidase (CO) activity, a metabolic marker of neuronal activity. Decreased hippocampal neurogenesis is thought to occur in depression and is decreased by 13-cis-RA. 5-HT is also a known regulator of hippocampal neurogenesis. Uncoupling of the dorsal raphe nuclei from the regions of the hippocampus by 13-cis-RA treatment may be the cause of, or a result from, the decreased neurogenesis. Although retinoids are known regulators of apoptosis, the uncoupling of the dorsal raphe nuclei from the hippocampal regions was not due to serotonergic cell loss. Interestingly, 13-cis-RA treated animals with the lowest CO activity in the dentate gyrus have the highest immobility in the tail suspension and forced swim tests. Ultimately, the effects of 13-cis-RA on the serotonergic and hippocampal systems might be inducing depression-related behaviors.
Beyond allostery: Catalytic regulation of a deoxyribozyme through an entropy-driven DNA amplifier
(Journal of Systems Chemistry, 2010-10-01) Eckhoff, Grace; Codrea, Vlad; Ellington, Andrew D.; Chen, Xi
The programmability and replicability of RNA and DNA have respectively enabled the design and selection of a number of allosteric ribozymes and deoxyribozymes. These catalysts have been adapted to function as signal transducers in biosensors and biochemical reaction networks both in vitro and in vivo. However, allosteric control of nucleic acid catalysts is currently limited by the fact that one molecule of effector (input) generally regulates at most one molecule of ribozyme or deoxyribozyme (output). In consequence, allosteric control is usually inefficient when the concentration of input molecules is low. In contrast, catalytic regulation of protein enzymes, as in protein phosphorylation cascades, generally allows one input molecule (e.g., one kinase molecule) to regulate multiple output molecules (e.g., kinase substrates). Achieving such catalytic signal amplification would also be of great utility for nucleic acid circuits. Here we show that allosteric regulation of nucleic acid enzymes can be coupled to signal amplification in an entropy-driven DNA circuit. In this circuit, kinetically trapped DNA logic gates are triggered by a specific sequence, and upon execution generate a peroxidase deoxyribozyme that converts a colorless substrate (ABTS) into a green product (ABTS•+). This scheme provides a new paradigm for the design of enzyme-free biosensors for point-of-care diagnostics.
Biological approaches to synthesis and assembly of semiconductor and metallic nanomaterials
(2005) Sweeney, Rozamond Yvonne; Iverson, Brent L.
The goal of this work is to use proteins, viruses, and whole organisms to direct the growth and assembly of semiconductor and metallic materials. The motivation for this work was to find a new way to build inorganic materials and devices with greater ease, more precise control, and smaller features than is possible with current synthetic methods. A biological method to efficiently synthesize large quantities of cadmium sulfide nanocrystals in the bacteria E. coli was discovered. The physical properties of the nanocrystals were characterized by electron microscopy and photoluminescence spectroscopy. Next, the genetic and physiological parameters that play a role in the synthesis of cellular nanocrystals were explored. In particular, a strain and growth phase dependence for E. coli nanocrystal formation was determined, indicating that the capacity for nanocrystal synthesis in E. coli is intrinsic and can be genetically controlled. This result is a first step towards understanding this mechanism of biologically-encoded nanomaterial synthesis, and it suggests the possibility of genetically engineering E. coli to produce nanocrystals with precise control over composition, size, and crystal type. Recently, it was discovered that filamentous viruses can be genetically engineered to direct the formation of semiconductor and magnetic nanowires. To follow-up on this project, a method for precisely directing the assembly of the viruses was developed. In order to begin ordering the viruses, the viral coat proteins were engineered to display a type of protein domain, called a leucine zipper, which can form non-covalent dimeric, trimeric, or tetrameric interactions with other leucine zippers. The leucine zipper, attached at the ends of the virus, caused individual viruses to adhere to each other end-to- end, producing one-and two-dimensional arrays. This method was also shown to be an effective way to alternate assembly of different types of viruses. By controlling the placement of the virus-templated nanowires from the bottom-up, the nanowires might become technologically useful for applications that require precise ordering, such as electronic and photonic circuits, sensors, or liquid crystal displays.
Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis
(Mobile DNA Journal, 2014-01-13) Enyeart, Peter J.; Mohr, Georg; Ellington, Andrew D.; Lambowitz, Alan M.
Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into ‘targetrons.’ Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and ‘cut-and-pastes’ (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.
Brain region gene expression responds discretely to chronic alcohol withdrawal with specific disruption of the hippocampus during intoxication
(2005) Berman, Ari Ethan; Bergeson, Susan E.
Alcoholism is a chronic, progressive and heritable disease that affects millions of Americans and costs the United States hundreds of billions of dollars per year in medical expenditures, property damage, and loss of productivity. Alcohol dependence is the result of long-lasting cellular and molecular changes in the brain that are initiated and maintained by the repeated ingestion of intoxicating amounts of alcohol. Withdrawal symptoms from alcohol occur when alcohol intake is reduced or halted and the brain enters a period of extended hyperactivity. Animal models for alcohol-related behaviors were previously developed and characterized in both mice and rats, including mouse strains that were particularly sensitive to alcohol withdrawal. Mice from a strain that is highly sensitive to withdrawal from alcohol, DBA/2J, were given a chronic dose of ethanol by inhalation and comparative microarray analysis was performed. A suite of microarray analysis software was written to facilitate the large amount of data collected from this experiment, and a robust web-based database system, the Alcohol Research Integrator, was developed to serve both as a storage and as a high-level analysis medium. Here we show that detectable gene expression changes occur in a discrete fashion between gross anatomical brain regions at various stages of withdrawal, and that the hippocampus shows a markedly greater level of gene expression change during intoxication than any of the other brain regions suggesting a particular vulnerability to the intoxicating effects of alcohol.
Bright/ARID3A contributes to chromatin accessibility of the immunoglobulin heavy chain enhancer
(Molecular Cancer, 2007-03-26) Lin, Danjuan; Ippolito, Gregory C.; Zong, Rui-Ting; Bryant, James; Koslovsky, Janet; Tucker, Philip
Bright/ARID3A is a nuclear matrix-associated transcription factor that stimulates immunoglobulin heavy chain (IgH) expression and Cyclin E1/E2F-dependent cell cycle progression. Bright positively activates IgH transcriptional initiation by binding to ATC-rich P sites within nuclear matrix attachment regions (MARs) flanking the IgH intronic enhancer (Eμ). Over-expression of Bright in cultured B cells was shown to correlate with DNase hypersensitivity of Eμ. We report here further efforts to analyze Bright-mediated Eμ enhancer activation within the physiological constraints of chromatin. A system was established in which VH promoter-driven in vitro transcription on chromatin- reconstituted templates was responsive to Eμ. Bright assisted in blocking the general repression caused by nucleosome assembly but was incapable of stimulating transcription from prebound nucleosome arrays. In vitro transcriptional derepression by Bright was enhanced on templates in which Eμ is flanked by MARs and was inhibited by competition with high affinity Bright binding (P2) sites. DNase hypersensitivity of chromatin-reconstituted Eμ was increased when prepackaged with B cell nuclear extract supplemented with Bright. These results identify Bright as a contributor to accessibility of the IgH enhancer.
Broad network-based predictability of Saccharomyces cerevisiae gene loss-of-function phenotypes
(Genome Biology, 2007-12-05) McGary, Kriston L.; Lee, Insuk; Marcotte, Edward M.
We demonstrate that loss-of-function yeast phenotypes are predictable by guilt-by-association in functional gene networks. Testing 1,102 loss-of-function phenotypes from genome-wide assays of yeast reveals predictability of diverse phenotypes, spanning cellular morphology, growth, metabolism, and quantitative cell shape features. We apply the method to extend a genome-wide screen by predicting, then verifying, genes whose disruption elongates yeast cells, and to predict human disease genes. To facilitate network-guided screens, a web server is available http://www.yeastnet.org webcite.
Buffering by gene duplicates: an analysis of molecular correlates and evolutionary conservation
(BMC Genomics, 2008-12-16) Hannay, Kevin; Marcotte, Edward M.; Vogel, Christine
Background: One mechanism to account for robustness against gene knockouts or knockdowns is through buffering by gene duplicates, but the extent and general correlates of this process in organisms is still a matter of debate. To reveal general trends of this process, we provide a comprehensive comparison of gene essentiality, duplication and buffering by duplicates across seven bacteria (Mycoplasma genitalium, Bacillus subtilis, Helicobacter pylori, Haemophilus influenzae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Escherichia coli), and four eukaryotes (Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (worm), Drosophila melanogaster (fly), Mus musculus (mouse)). -- Results: In nine of the eleven organisms, duplicates significantly increase chances of survival upon gene deletion (P-value ≤ 0.05), but only by up to 13%. Given that duplicates make up to 80% of eukaryotic genomes, the small contribution is surprising and points to dominant roles of other buffering processes, such as alternative metabolic pathways. The buffering capacity of duplicates appears to be independent of the degree of gene essentiality and tends to be higher for genes with high expression levels. For example, buffering capacity increases to 23% amongst highly expressed genes in E. coli. Sequence similarity and the number of duplicates per gene are weak predictors of the duplicate's buffering capacity. In a case study we show that buffering gene duplicates in yeast and worm are somewhat more similar in their functions than non-buffering duplicates and have increased transcriptional and translational activity. -- Conclusion: In sum, the extent of gene essentiality and buffering by duplicates is not conserved across organisms and does not correlate with the organisms' apparent complexity. This heterogeneity goes beyond what would be expected from differences in experimental approaches alone. Buffering by duplicates contributes to robustness in several organisms, but to a small extent – and the relatively large amount of buffering by duplicates observed in yeast and worm may be largely specific to these organisms. Thus, the only common factor of buffering by duplicates between different organisms may be the by-product of duplicate retention due to demands of high dosage.
The catalytic mechanism of dimethylarginine dimethylaminohydrolase (DDAH) from pseudomonas aeruginosa
(2006) Stone, Everett Monroe; Fast, Walter
Dimethylarginine dimethylaminohydrolase (DDAH) catalyzes the hydrolysis of Nw–methyl–L–arginine (NMMA) and Nw,Nw –methyl–L–arginine (ADMA) to L-citrulline and methylamine or dimethylamine, respectively. ADMA and NMMA are endogenous inhibitors of nitric oxide synthase (NOS) in mammals. DDAH therefore partially regulates NOS activity, making it an attractive therapeutic target in disease states involving overproduction of nitric oxide. Understanding the mechanism of DDAH is important to inhibitor design and elucidating its physiological function. DDAH is a member of the amidinotransferase superfamily, and has conserved active–site residues including cysteine, histidine, and glutamate/aspartate that are integral to catalysis. In DDAH from Pseudomonas aeruginosa, the active–site Cys249 is activated as a nucleophile upon binding substrate, and forms a covalent intermediate concomitant with loss of the alkylamine leaving group. The active–site His162 has a dual role, first as a general acid in protonating the alkylamine leaving group and second as a general base in generating a hydroxide for attack on the covalent intermediate. The active–site Glu114 is essential for properly orienting and ionizing His162. The use of a substrate analog, S–methyl-L-thiocitrulline (SMTC), enabled development of a new method of continuously monitoring DDAH activity, allowing facile screening of inhibitors. Using this method, a haloacetamidine was identified as an active–site directed inactivator motif for DDAH, and other members of the amidinotransferase superfamily.
Cbl-b: its role of expression and regulation in T-lymphocyte activation and ageing
(2007) Xu, Zhun, 1973-; Jolly, Christopher A.
The aging process is strongly associated with decreased activity in the immune system. Dysregulation of T-lymphocyte function, such as reduced proliferation, is one problem faced by most elder people, which prevents them from successfully dealing with exogenous pathogens. Effective regulation of T-lymphocyte activity depends on the proper and prompt transduction of both positive and negative signals within Tlymphocytes and reflects the balance between positive and negative effects. Decline of positive signaling in aging has been studied and reported, while mechanisms concerning up-regulation of negative signaling with age and its role in immune senescence are still unclear. Cbl-b, an E3 ubiquitin ligase, was studied by our lab since it regulates the ubiquitin process, a protein modification process that has suppressive effects on signaling pathways. We first determined the reaction of Cbl-b to different stimuli in young rat splenic T-lymphocytes, and showed that there is a decrease in Cbl-b protein expression upon CD28 stimulation and such protein degradation is proteasome-dependent only. We also showed the mechanism of Cbl-b expression regulation involves the intracellular movement of Nedd4 toward Cbl-b and an up-regulation of Nedd4 expression. Then we proved that in old splenic T-lymphocytes, decreased proteasome activity was unable to down-regulate the Cbl-b protein. High levels of Cbl-b in old T-lymphocytes are functional in preventing PI3K activity and are associated with reduced T-lymphocyte proliferation upon regular stimulation. T-lymphocytes from old Cbl-b knock-out mice show similar proliferative reaction to CD3 stimulation as T-lymphocytes from young wild-type, which establishes the causeeffect relationship between sustained Cbl-b expression and decreased T-lymphocyte proliferation. In summary, these data suggest a unique role of Cbl-b in regulating Tlymphocyte signal transduction and provide critical preliminary data for extending Cbl-b studies into other fields, such as carcinogenesis.
Characterization of factors involved in 3' to 5' mRNA degradation in yeast
(2005) Wang, Lingna; Johnson, Arlen W.
The pathways for eukaryotic mRNA degradation are composed of numerous interconnected elements. I have characterized the general roles of factors involved in 3’ to 5’ mRNA degradation pathway in the budding yeast Saccharomyces cerevisiae. The Ski2/3/8 complex [composed of a DEVH ATPase—Ski2p, a TPR (Tetratricopeptide Repeat) protein—Ski3p and a WD protein—Ski8p] and Ski7p (a putative GTPase) are essential to 3’ mRNA degradation. To better understand their role in mRNA decay, first, I explored the domain interactions within the Ski2/3/8 complex and between the Ski2/3/8 complex and Ski7p using a directed two hybrid approach combined with IP (immunoprecipitation) experiments. A model that describes all the interactions identified is presented. Since Ski2p belongs to the DEVH RNA helicase family, I then demonstrated the biochemical activity of the Ski2/3/8 complex in vitro. My results indicate that the Ski2/3/8 is a hetrotrimeric complex with the stoichiometry of 1:1:1. The complex contains ATPase activity, which is specific to Ski2p. The Ski2/3/8 complex also possesses 3’ to 5’ RNA helicase activity. The duplex unwinding activity is intrinsic to Ski2p and requires the ATPase activity of Ski2p. More importantly, the helicase acitivity of Ski2p is stimulated by the presence of a short poly(A) tail (A12). Interestingly, mutations in Ski2p changing DEVH to AEVA cause this protein to be dominant negative. The dominant negative phenotype is caused by outcompeting wildtype Ski2p for incorporation into the Ski2/3/8 complex and forming a large inactive complex with the exosome. To better understand the role of GTPase Ski7p in 3’ mRNA decay, a dominant negative screen of SKI7 was carried out. Further characterization of the identified dominant negative mutants of SKI7 revealed that these mutants altered the interaction with either the Ski2/3/8 complex or the exosome, supporting the idea that Ski7p functions as a mediator between the Ski2/3/8 complex and the exosome in 3’ to 5’ mRNA degradation.
Characterization of Herc5: the major ligase for ISG15, an antiviral ubiquitin-like protein
(2007-08) Dastur, Anahita R., 1975-; Huibregtse, Jon M.
Human ISG15 is a 17 kDa ubiquitin-like protein (Ubl) that is induced by type I interferons (interferons [alpha] and [beta]) and plays a role in antiviral responses. ISG15 is conjugated via its C-terminus to more than 150 cellular proteins, and like ubiquitin, an E1-E2-E3 enzymatic cascade is required for conjugation. Ube1L and UbcH8 were previously identified as the E1 and E2 enzymes for this pathway. My experiments identified Herc5, a HECT domain E3, as the major ligase for ISG15. Like ISG15, Ube1L, and UbcH8, expression of Herc5 is transcriptionally induced by type I interferons. siRNAs against Herc5 abrogated ISG15 conjugation to the vast majority of target proteins in interferon-treated cells. Wild type Herc5, but not the catalytically inactive C994A mutant, supported conjugation of ISG15 in non-interferon-treated cells co-transfected with Ube1L, UbcH8 and ISG15. IQGAP1, a scaffold protein, was identified as another essential component of the ISG15 system. IQGAP1 was discovered to interact with Herc5, and this interaction was mediated by the C-terminal domain of IQGAP1 and the N-terminal RCC1-like repeats of Herc5. IQGAP1 was required for auto-conjugation of ISG15 to Herc5, and I propose a model where IQGAP1 functions, at least in part, by relieving an auto-inhibitory conformation of Herc5. Thus, I have identified two factors that are critical for ISG15 conjugation and my discoveries have increased our understanding of the ISG15 pathway. Identification and characterization of the conjugation apparatus will aid in establishing an in vitro biochemical system for ISG15 conjugation, which in turn, will be important to decipher the biological function of ISG15 modification.
Characterization of metastasis regulators in human breast cancer: implications for tumor suppressor PTEN and the Rho family of small GTPases
(2005) Baugher, Paige Jennette; Dharmawardane, Surangani
Cancer metastasis is a multi-faceted process requiring the disregualtion of numerous signaling pathways, including those associated with cell adhesion and motility. Recent data indicates strongly that growth at a primary tumor site and growth at a metastatic site differ by the expression and/or context-dependent function of the metastasis regulator, and that a wide variety of signaling pathways are affected. PTEN (phosphatase and tensin homologue deleted on chromosome ten) then becomes an attractive candidate for a metastasis suppressor, based on its ability to negatively regulate numerous pathways involved in cell survival, cell proliferation, and cell motility. Conversely, the Rho family of small GTPases have become attractive candidates as contributors to metastasis. Rho GTPases regulate numerous signaling pathways involved in cell survival, cell proliferation and cell motility, but they function to enhance these processes instead of inhibiting them. Data presented here demonstrates the ability of PTEN to negatively regulate motility in human metastatic breast cancer cells without causing the cells to undergo apoptosis. PTEN is localized in stimulated cells away from the leading edge, which displaces it from sites of active motility signaling and prevents it from inhibiting these processes. Furthermore, ectopic PTEN expression is shown to downregulate phosphoinositol (3,4,5) triphosphate (PIP3), expression. Therefore, PTEN could be acting as a metastasis suppressor in human breast cancer. Data presented here also demonstrate the ability of the Rac subfamily of Rho GTPases to enhance metastatic properties and contribute to metastasis. Increased Rac activity was shown to correlate with increased metastatic potential in a panel of metastatic human breast cancer cell variants. When activated Rac1 or Rac3 was expressed stably in the least metastatic variant, either isoform was found to enhance adhesion, migration, and invasion in vitro, as well as contribute to pulmonary metastasis in the nude mouse model of experimental metastasis. Conversely, when dominant negative Rac1 or Rac3 was expressed in the most metastatic variant, either isoform was found to decrease adhesion, migration, and invasion in vitro, as well as block pulmonary metastasis in vivo. Therefore, Rac1 and/or Rac3 are found to act as metastasis regulators by negatively regulating metastatic human breast cancer progression.
Characterization of S. flexneri DegP
(2003) Purdy, Georgiana Elizabeth; Payne, Shelley M.
Shigella are Gram-negative facultative intracellular pathogens that cause bacillary dysentery. An important aspect of Shigella pathogenesis is the ability of the bacteria to invade intestinal epithelial cells, multiply within the eukaryotic cell cytoplasm, and spread to adjacent cells by the polymerization of actin. An S. flexneri degP mutant was isolated in a genetic screen for mutants that invaded Henle cells but did not form wild type plaques in Henle cell monolayers (51, 52). The purpose of this study is the characterization of an S flexneri degP mutant. DegP is essential for virulence in other bacterial pathogens, therefore the role of DegP in S. flexneri virulence was investigated. Studies with the S. flexneri degP mutant indicated that DegP may be important for several stages of Shigella infection including transit through the stomach, intercellular spread and bacterial clearance by the host immune response. In addition, due to the conserved function of DegP in bacterial extracytoplasmic stress response, investigations also were undertaken into the role of S. flexneri DegP in various environmental conditions.