Recent Submissions

  • Autism spectrum disorder traits in SLC9A9 knock-out mice

    Faraone, Stephen; Yang, Lina (2014)
    utism spectrum disorders (ASDs) are a group of neurodevelopmental disorders which begin in childhood and persist into adulthood. They cause lifelong impairments and are associated with substantial burdens to patients, families and society. Genetic studies have implicated the sodium/proton exchanger (NHE) nine gene, SLC9A9, to ASDs and attention-deficit/hyperactivity disorder (ADHD). SLC9A9 encodes, NHE9, a membrane protein of the late recycling endosomes. The recycling endosome plays an important role in synapse development and plasticity by regulating the trafficking of membrane neurotransmitter receptors and transporters. Here we tested the hypothesis thatSLC9A9 knock-out (KO) mice would show ADHD-like and ASD-like traits. Ultrasonic vocalization recording showed that SLC9A9 KO mice emitted fewer calls and had shorter call durations, which suggest communication impairment. SLC9A9 KO mice lacked a preference for social novelty, but did not show deficits in social approach; SLC9A9 KO mice spent more time self-grooming, an indicator for restricted and repetitive behavior. We did not observe hyperactivity or other behavior impairments which are commonly comorbid with ASDs in human, such as anxiety-like behavior. Our study is the first animal behavior study that links SLC9A9 to ASDs. By eliminating NHE9 activity, it provides strong evidence that lack of SLC9A9 leads to ASD-like behaviors in mice and provides the field with a new mouse model of ASDs.
  • FH2-dependent Localization of FHOD Formins in the Sarcomere

    Blystone, Scott; Hamilton, Elisabeth (2016)
    Formins are a class of actin nucleating factors containing a highly-conserved FH2 domain, which binds actin. There are 15 mammalian formin proteins in seven sub-families that have been found to nucleate, cap, sever, bundle, and polymerize linear actin filaments. An expression analysis of all 15 human formins across 22 different cell and tissue types showed high levels of expression for one formin sub-family in striated muscle: FHOD. While FHOD1 is highly expressed across many cell and tissue types, FHOD3 is only found at comparatively high levels in striated muscle cells. The mature structure of skeletal muscle is a very recognizable periodic repetition of filaments that allows muscles to contract. The sarcomere is the smallest contractile unit of skeletal muscle, with the two main filaments, the thick filament containing myosin, and the thin filament containing actin moving back and forth via the interaction of myosin heads with the thin filament, which allows muscles to contract. While the molecular functioning and the mature structure of skeletal muscle is well understood, the exact mechanism by which the sarcomere is assembled, and specifically, how the actin-core of the thin filament is formed and incorporated into the thin filament remains unknown. The high levels of expression of FHOD formins in striated muscle, combined with their ability to interact with filamentous actin warranted a closer look at FHOD1 and FHOD3 in the sarcomere. In this study we found that FHOD1 and FHOD3 have distinct sarcomeric localizations in C2C12 cells. FHOD1 localizes to the barbed end of the actin filament at the Z-disk and FHOD3 localizes to the pointed end of the actin filament near the center of the sarcomere. Full-length cDNA constructs for FHOD1 and FHOD3 were introduced into skeletal muscle cells, and we were able to recreate the endogenous localization of FHOD3 with the exogenous cDNA. The FHOD1 cDNA localized not to the barbed end of the actin filament, as endogenous FHOD1 does, but instead to the pointed end of the actin filament, where endogenous and exogenous FHOD3 were found. We hypothesized that FHOD binding to actin is dependent upon two highly conserved actin-binding sites in the FH2 domain. Mutations of the actin-binding residues in the FH2 domain impaired the actin-binding ability of both exogenous FHOD1 and FHOD3 and showed that the localization of FHOD1 and FHOD3 in the sarcomere is actin-binding dependent.
  • Functional Studies of Tumor Suppressor ECRG2 in the Regulation of Cancer Cell Death and Drug Resistance

    Huang, Ying; Lucchesi, Chris (2015)
    Esophageal Cancer Related Gene 2 (ECRG2) is a newer tumor suppressor whose mRNAhas previously been shown to be decreased in multiple human malignanceswhencompared to normal/adjacent tissues.Of importance, ECRG2 has previously been revealedto possess tumor suppressive attributes,including the ability to induce cell death in cancer cells. However, how ECRG2 is able to activate the apoptotic machinery has yet to be elucidated. In the present study,we highlight multiple angles that ECRG2 leverages in order to sensitize cancer cells to apoptosis. Moreover, we report for the first time,that ECRG2 protein expression in lung cancer patientsamples is lost/decreased in upwards of 90% ofthecancer tissues evaluatedcompared to normal tissue. Additionally, a single somatic point mutant found in patient tumor samples, V30E, was shown to lose tumor suppressive abilities and acquired resistance against multiple anticancer drugs. Our results demonstrate that ECRG2is upregulated in response to DNA damage, andis capable of inducing the activation of both caspase cascades (intrinsic and extrinsic) leadingto cancer cell death. We further illustratedthat ECRG2-mediated cell deathwasattributed to a reduction in the levels of apoptosis inhibitor, X chromosome-linked inhibitor of apoptosis protein (XIAP). ECRG2 was revealedto regulate XIAP protein levels via RNA-binding protein,human antigen R(HuR).We further highlight that ECRG2 causes increased HuR ubiquitination,subsequently leading to its degradation. Thus, we demonstrate that ECRG2 sensitizescancer cells to apoptosis through the downregulation of HuR, and consequent downregulation of XIAP. Next, we have identified ECRG2 as a potent positive regulator of death receptor 5 (DR5) gene expression.ECRG2-mediated upregulation of DR5 was shown to be facilitated through the upregulation of tumor suppressor p53 and transcription factors ATF3 and NFⱪB. Together, in a cooperative fashion, increased levelsof p53, ATF3 and NFⱪB stimulateDR5 gene expression. Contrastingly, silencing of ECRG2 not only decreased the levels of NFⱪB and DR5, but also resulted in decreased cancer cell sensitivity to genotoxic stressandTRAIL treatment. Collectively,our work establishes that ECRG2 is capable of inducing apoptosis in cancer cells by increasing the expression of pro-apoptotic proteins, while also negatively influencing anti-apoptotic proteins.Further, the loss of ECRG2’stumor suppressive abilities, as wehaveshownby the loss of ECRG2in lung patient tumor samples,and through the somatic point mutantV30E, illuminates possible mechanismsin which cancer cells can acquiremultiple drug resistance.
  • DISSECTING THE ROLE OF YEAST RIM8 IN THEINTERNALIZATION OF THE PLASMA MEMBRANE PROTON PUMPPMA1

    Kane, Patricia; Shoniwa, Makandiwana (2013)
    The vacuolar A-TPase (V-ATPase) is a proton pump that is found ubiquitiously throughout the cells. It uses the hydrolysis of ATP to transport protons across membranes, thereby maintaining homeostatic pH. pH control in the cells of an organism is vital, a disturbance in cellular pH may be lethal. The maintenance of homeostatic pH within the cell appears to be a result of the interplay between V-ATPasesandproton exporters. In yeast and plants, the major proton exporter isthe plasma membrane proton exporter, Pma1. Pma1 is the transporter that is primarily involved in themaintenance of cytosolic pH. In cells in which the function of V-ATPase has been compromised (vma mutants) Pma1 is partially mislocalized.It is known thatmembrane transporterslacking the PY motifare endocytosed via the action of an Arrestin Related Trafficking (ART) protein, which translocates an E3 ligase into close proximity with the transporter, so as to allow for the ubiquitination of the transporter. Rim 8 is the ART protein (adaptor) that has been linked to the endocytosis of Pma1, along with E3 Ubiquitin ligase Rsp5. It is of interest to this project that Rim8 is well studied in its role as an adaptor in the alkaline ambient pH pathway. We thus propose that there may be crosstalk between the ambient pH pathway and the pathway that leads to the internalization of Pma1. Therefore, in this body of work we seek to find other players that may be involved in the Pma1 pathway, as well as to elucidate theareas of interaction between Rim8 and Pma1. Ourfinal goal isbringing a better understanding ofthe pathway that leads to the endocytosis of Pma1. To answer the question posed in this work we monitored the growth phenotype and the localization of Pma1 indouble mutants lacking both V-ATPase subunits and key players in the ambient pH pathway. In addition, we looked to see which cytosolically exposed terminal of Pma1 may be involved in theinteraction with Rim8. In yet another experiment, we mutatedRim8 so as to find which areas of the adaptor werevital for Pma1 internalization.Our results showed that other players tested in the Rim pathway(vma2∆rim20∆and vma2∆vps23∆)werenot required for Pma1 internalization. In addition we observed that mutations in Rim8 that compromise its function in the Rim pathway still allow Pma1 internalization, even though they show synthetic growth phenotypes with vma2∆ mutations. Two-hybrid assay could not detect thesites of interaction between Rim8 and Pma1 and newstrategies will be employed to determine these sites. Changes in electrophoretic mobility of Rim8 suggested that Rim8 undergoes posttranslational modifications, and showed differences in vma2∆mutants and WT mutants.
  • UNDERSTANDING THE CELLULAR AND MOLECULAR MECHANISMS OF CEREBRAL CAVERNOUS MALFORMATION 3 (CCM3)

    Howell, Brian; Mansaray-Storms, Zainab Y. (2016)
    Cerebral cavernous malformation 3 (CCM3) is one of three genes which when mutated plays a role in the neurovascular disease, cerebral cavernous malformation. Through a number of diverse binding partners, CCM3 plays a critical role in modulating several processes including cell survival, migration and vascular development. However, how CCM3 regulates many of these pathways remains unclear. An interaction with Stk25, a serine/threonine kinase with roles in cell polarity suggests CCM3 might play a role in the functions of Stk25. Here we characterize a role for CCM3 in cellular polarity. We identify a function for CCM3 in epithelial polarity through its association with and regulation of the conserved LKB1 signaling pathway. We find that CCM3 associates with STRADα, the regulatory pseudokinase of the LKB1 complex, and is necessary for LKB1-mediated cell polarization. To determine whether this novel association of CCM3 with STRADα and LKB1 pathway plays a role in CCM3-phenotype in endothelial cells, we analyzed the gene expression profiles of endothelial cells deficient in CCM3 protein. We identified changes in gene expression induced by CCM3 knockdown, particularly a significant downregulation in expression of cell adhesion molecules, a dysregulation of extracellular matrix signaling and an activation of p53 signaling pathway. This work defines a novel regulatory role for CCM3 in epithelial cell polarity and provides preliminary insights into downstream signaling pathways affected by the reduction of CCM3 in endothelial cells with potential impact in CCM disease pathogenesis.
  • MITOCHONDRIAL PROTEINS AS TUMOR MARKERS AND ANTI-CANCER DRUG TARGETS

    Sheikh, Saeed; Babbar, Mansi (2017)
    Cancer is a major cause of morbidity and mortality. Identification and characterization of novel biomarkers are expected to facilitate early diagnosis and improve prognosis of human malignancies. Increasing number of studies have linked tumor progression with metabolic reprogramming. However, the players involved are not fully discovered. Therefore, understanding the cancer cell plasticity may offer a successful approach for an anti-cancer strategy. In this regard, we report the functional characterization of Coiled-coil Helix Tumor and Metabolism 1 (CHTM1) and KM1 as important regulator of cancer cell metabolism.CHTM1 is localized in cytosol and mitochondrial inter-membrane spaceand regulates mitochondrial activity. Our results demonstrate that MIA40 appears to alterCHTM1 mitochondrial localizationand stability. Further, CHTM1 cysteineresiduesinvolved in CHTM1 folding modulatescellular distributionof CHTM1. Importantly, alterations in CHTM1 expression in cancer cells affect mitochondrial activity. Given thatmitochondria play an important role in cellular response to nutrient stress, we sought to analyze the role of CHTM1 in glucose/glutamine-deprived conditions. Wehave found thatCHTM1 deficiency enhancescancer cell sensitivityto glucose/glutamine starvation and metformin treatment. Additionally, increased sensitivity of CHTM1-deficient cells to metabolic stress could be in part due to inability to activate fatty acid oxidation. Further, targeting CHTM1 expression in cancer cells reduce fatty acid oxidation causing decrease in substrate availability under metabolic stress conditions. This can explain the increase in autophagy and protein catabolism in CHTM1-deficient cancer cells under metabolic stress conditions. Mechanistic studies suggest that CHTM1-mediated alterations in cancer cell metabolism under stress conditions involve modulation of PGC1 alpha-CREB-PKC signaling.We further demonstrate that under metabolic stress, CHTM1 deficiency activates p38-AIF1pathway leading to increased cell death. CHTM1 negatively regulates p38 and interacts with AIF1 alteringAIF1release frommitochondria under metabolic stress conditions.These findings are highly significant because alterations in cancer cell metabolism are linked to pathogenesis of cancer. Most importantly, multiple human malignancies associated with breast, colon and lung tissuesshow increase in CHTM1 expression. CHTM1 appears to be a high value tumor marker, that has the potential tofacilitate earlydiagnosis of human malignancies and could also serve as a target to develop novel therapeutics to manage human malignancies. In the second part of this manuscript, we report the characterization of a novel protein temporarily named as KM1. Our results indicate that KM1 is localized inthemitochondrial inner membrane and regulates mitochondrial activity. Metabolic stress-induced increased cell death is noted in KM1 knockout cancer cells, a finding consistent with the defective mitochondria in KM1-deficient cells. Our results further demonstrate that under metabolic stress KM1 regulates mitochondrial-mediated cell death. Most importantly, KM1 levels are upregulated in breast and lung cancer tissues.Collectively, our results suggest that CHTM1 and KM1 are novel proteins and are involved in regulating cancer cell metabolism.
  • IDENTIFICATION OF p53-MEDIATED NEUROGENOMIC RESPONSES TO ETHANOL USING IN VIVO AND IN VITRO MODELS OF FETAL ALCOHOL SPECTRUM DISORDER

    Camargo, Maria (2016)
    Fetal Alcohol Spectrum Disorder (FASD) is a serious public health concern affecting 3.6% of the US population. One avenue to achieve a decrease in the prevalence of FASD is for scientific research to identify cellular mechanisms of action of imbibed alcohol and propose solutions to treat or prevent the damage done. Here we present our investigation into the molecular consequences of ethanol exposure in mouse brain cells and mouse neural stem cell cultures. Specifically, we tested the hypothesis that p53 mediates the neurogenomic response to ethanol exposure in brain cells in the somatosensory cortex, hippocampus and neural stem cells. p53 is a versatile transcription factor well known for inducing cell death in cancer cells. We identified the apoptosis pathway as being changed in a p53-related manner only in the CA1 subregion of the hippocampus, based on expression changes in Casp2, Cdk1, and Stat1. Overall, the regions interrogated revealed that p53’s cellular response is heterogeneous. In the somatosensory cortex and hippocampus a subset of gene expression changes occurred depending on both ethanol exposure and the presence of p53: Ephb1in layer 2/3; Ctgf in layer 5; Camk1 in layer 6; Cdk1, Casp2, Cdk1, and Stat1 in the CA1; and Camk1 in the DG. In regards to the specific mRNAs that changed, they differed in the brain regions and cell cultures, but we did observe that neuronal and developmental genes were the most significantly changed upon ethanol exposure. In addition, we also identified that the category of genes whose methylation pattern was changed after ethanol exposure are related to basic neuronal functions. Neural cells also appeared to be engaged in a challenging response to ethanol because DNA repair proteins Ercc1, Hus1, and Rad51 alter their DNA binding after ethanol exposure. In addition, we identified that p53 transcription factor changes its DNA binding in response to ethanol exposure. In conclusion, we identified that neural p53 signaling is measurably perturbed by ethanol exposure.
  • The Chemosensory-­Related Consequences of Fetal Ethanol or Fetal Nicotine Exposure: Their Contribution to Postnatal Nicotine Acceptance

    Youngentob, Steven; MANTELLA, NICOLE (2015)
    Human studies demonstrate a predictive association between gestational exposure to alcohol or nicotine and the probabilityoflater nicotine dependence.The flavor qualitiesof both drugsare known to influencetheir earlyacceptance and they share the perceptual attributesof an aversive odor, bitter taste and oral irritation.This dissertationexamined whether there are chemosensory-­‐related consequences offetal: (1) alcohol exposurethat contribute toenhanced nicotine acceptance; or (2)nicotine exposure that also enhances acceptance. The study rationale was drivenby overlappingliteraturesrelated to: (1) the relationship between gestational exposurewith chemosensory stimuli and their postnatal acceptance; (2) lessons learned from prenatal alcohol exposure and its postnatal consequences; and (3) perceptual commonalities between the flavor of alcohol and nicotine.Alcohol studies: rats were alcohol-­‐exposed during gestationvia the dams’ liquid diet. Control damsreceived ad libaccessto an iso-­‐caloric, iso-­‐nutritive diet. Nicotine studies: dams’ were implanted with a mini-­‐osmotic pump containing nicotine.Control animals received either vehicle only or no pump. Behaviorally, we found that fetal alcohol exposed adolescent rats showed anenhanced nicotine odor
  • Targetingof PIM1 KinaseinMyeloproliferative NeoplasmsInduced by JAK2V617F

    Mohi, Golam; Stuver, Matthew (2017)
    Myeloproliferative neoplasms (MPNs) arestem cell-derivedblood disorders. The most common mutation found in MPN patients is the JAK2V617Fmutation. JAK2 is anon-receptor tyrosine kinase involved in STAT signaling. The JAK2V617F mutation is asingle amino acid substitution of a phenylalanine for valine, whichcauses JAK2 to be constitutively activated. This mutation can cause ahematopoietic transformation. Eventuallythis transformationcan lead to the development of one of thethree different Philadelphia-negative MPN diseases: Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF). The JAK2V617F mutationhas been identified in 95% PVpatients,and 50-60% ofETand PMF patients.A JAK1/2 inhibitor (ruxolitinib) has been approved for MF and PV patients and,though it provides initial benefits, it is not effective enough to causelong-termremission in patients. This creates a critical need to identify new therapeutic targets for MPN patients. We found that PIM1 levels were significantly increased inMPN patients, as well asour JAK2V617F mouse modelof MPN.We observedthatknockdown of PIM1 caused a significant decrease in proliferationof JAK2V617F expressing cells. We also found that PIM1 knockdownhad no effect on the proliferation of hematopoietic cells not expressing JAK2V617F, leading us to believe PIM1 is only required in JAK2V617F mediated proliferation. Pharmacological inhibition of PIM kinases,using TP-3654,(kindly provided by Tolero pharmaceuticals)also led to a significant decrease in proliferation of JAK2V617F-expressing cells, but had no effect on cellslacking the mutation. We also found thatthePIM inhibitor,TP-3654,workssynergistically with ruxolitinibto achieve an even greater decrease in proliferation. We found that using the combination of ruxolitiniband TP-3654,we could use both drugs at lower concentrations andachieve an even greater decrease in proliferation and an increase apoptosis. Furthermore,we found that inhibition of PIM kinasesusing TP-3654can resensitize ruxolitinib-resistant cells to ruxolitinibtreatment.These important findingsshow that PIM1 plays animportantrolein the proliferation of hematopoietic cells expressing the JAK2V617F mutation, but is dispensable for the maintenance of cells lacking the mutation. We also found that targeting PIM kinases with TP-3654,significantly decreasedthe proliferation, and increaseapoptosisactivationof JAK2V617Fexpressing cells. We also showedthat TP-3654 and ruxolitinibcan work synergistically. Lastly, we showed that inhibition of PIM kinases,using TP-3654,caused ruxolitinib-resistant cells tobecome resensitized toruxolitinib. These findings helpedus come to the conclusionthatPIM1 kinase, is an importanttherapeutictargetin JAK2V617F-induced MPNs.
  • Domain swapping, fragment complementation, and self-assembly in engineered chimeric proteins.

    Loh, Stewart; Craft, Matthew (2014)
    Domain swapping is a form of protein oligomerization in which two or more identical proteins reciprocally exchange parts of their tertiary structure. The structure of the domain swapped proteins is identical to the structure formed by the monomer except for the hinge region linking the two domains. Domain swapping provides the cell with a way to control complex assembly, alter enzyme kinetics and specificity. Domain swapping can also be used to reconstitute enzyme function or as a form of molecular recognition. Only a small number of proteins are known domain swap, and the forces behind domain swapping are not well understood. Much more need to be understood abouthow and why proteins domain swap, before it would be possible to reliably engineer proteins to do so. In an effort to understand the thermodynamic forces that drive domain swapping, the goal of this project was to induce domain swapping and investigate the effects different hinge regions have on an otherwise identical domain swapped structure. To accomplish this we inserted ubiquitin (Ub) into five surface loops of ribose binding protein (RBP), a protein that does not naturally domain swap. The presenceof ubiquitin puts conformational strain on RBP and vice versa, where the folding of one causes the other to unfold. The entropic penalty for having unfolded domains can be relieved by domain swapping, allowing all protein domains to be folded. Using gelfiltration and circular dichroism we determined that our RBP-Ub (RU) fusion proteins domain swap. This domain swapping is dependent upon the conformational strain caused by a folded Ub, and can be reversed by the addition of a flexible glycine linker. Using our RU system we provide the first evidence that proteins with non-identical hinge regions can domain swap to form stable, functional oligomers. Finally we present a physical model that explains the ability of different RU’s to domain swap, which provides at least some of the criteria required of domain swapping proteins.
  • The Formin FMNL1 Contributes to the Macrophage Inflammatory Response by Regulating Podosome-dependent Adhesion and Migration.

    Blystone, Scott; Miller, Matthew (2015)
    Macrophages are indispensible white blood cells (leukocytes) that contribute to both the innate and adaptive immune response. They are crucial for resolving inflammatory events by clearing pathogens and cellular debris, in addition to promoting wound repair. Macrophages are derived from peripherally circulating monocytes, which after stimulation undergo diapedesis from the vasculature into the underlying complex extracellular matrix, where they can become fully differentiated macrophages and migrate to inflammatory loci. Tissues also contain residential populations of macrophages that aid in immediate immune responses and maintain tissue homeostasis. Conversely, unwarranted macrophage activation largely contributes to the onset and progression of inflammatory diseases, such as atherosclerosis and rheumatoid arthritis, in addition to aiding cancer metastasis and facilitating organ transplant rejection. In order for macrophages to effectively resolve inflammatory events or contribute to disease pathology, they must be able to undergo directional migration, which is mediated by integrin-dependent adhesion complexes termed podosomes. Macrophage podosomes are the most prominent structure of the macrophage actin cytoskeleton, containing a pillar-like core of dense filamentous actin that is tethered to the cortical actincytoskeleton via radial actin filaments. Podosomes also contain a variety of proteins that are circumferentially arranged orassociated with the core, and thatare involved in signaling, linking, and scaffolding,as well as modulating the actin cytoskeleton.Historically, our lab has been interested in leukocyte integrin biology and understanding how these receptors mediate adhesion and migration through complex extracellular matrices. Previous studies in our lab demonstrated the novel podosomal association of an actin modulating protein with the ability to processively elongate unbranched linear actin filaments. Subsequent studies determined this protein to be the formin FMNL1, which is predominantly expressed in hematopoietic cells. Consequently, we further revealed that FMNL1 localizes to the apex of the dense actin core, and is required for podosome stability and macrophage adhesion.The work described in this dissertation has greatly expanded on these findings, demonstrating for the first time that primary macrophage migration is dependent on the formin FMNL1. Utilizing in vitro and in vivotechniques with aid of a novel conditional murine FMNL1 KO, we have observed that macrophage podosome formation, migration, and tissue distribution are dependent on FMNL1. Additionally, we have indicated that FMNL1 is required for embryonic development. Remarkably, our findings also suggest that FMNL1-dependent macrophage migration and podosome localization rely on the specific isoform FMNL1γ. Foremost, we have demonstrated that barbed end binding by the FMNL1γ FH2 domain is dispensable for its cellularfunction in macrophages, which has not been previously shown for any other cellular formin function. Thus, these findings, in addition to current formin knowledge, have allowed us develop a working model for FMNL1 function at macrophage podosomes. This work has distinguished FMNL1 as a unique therapeutic target to restrict macrophage migration that contributes to macrophage-mediated diseases. Furthermore, this could translate to treatment of certain cancers, since FMNL1 has been suggested to promote leukemic cell migration.
  • Reconstitution and Characterization of RNA Polymerase I Upstream Activating Factor.

    Knutson, Bruce; Smith, Marissa (2018)
    RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and one of the most critical steps in ribosome biosynthesis. Pol I transcription initiation is coordinated by four Pol I factors that include the Upstream Activating Factor (UAF), TATA-binding protein (TBP), Core Factor (CF), and Rrn3. These factors work together to recruit Pol I to the rDNApromoter and to initiate transcription.UAF is a six-subunit complex composed of Rrn9, Rrn5, Uaf30, Rrn10, and histones H3 and H4.To investigate the importance of each UAF subunit in UAF complex formation and complex integrity, we developed a recombinant Escherichia coli-based system to coexpress and purify transcriptionally active UAF complex. Here, we found that no single subunit is required for UAF assembly, including histones H3 and H4. We also demonstrate that histone H3 is able to interact with each UAF-specific subunit. Last, wedetermined the stoichiometry of the subunits of the UAF complex, revealing there are two copies of histoneH3 and one copy of the remaining UAF subunits, including histone H4. Together, our results provide a new model suggesting that UAF contains a hybrid H3–H4 tetramer-like subcomplex.The results from this thesiswill help to reveal key mechanisms in Pol Itranscription activation.
  • Characterizing the Role of the Epsilon Subunit in Regulation of the Escherichia coli ATP Synthase.

    Duncan, Thomas; Shah, Naman (2015)
    The F-type ATP synthase is a rotary nanomotor central to cellular energy metabolism in almost all living organisms. In bacteria, the enzyme also plays a role in nutrient uptake and pH regulation underlining its importance. All ATP synthases can be inhibited by ADP, whereas in bacteria, the enzyme is alsoautoinhibitedbyits ε subunit. The inhibition involves a drastic conformationa l change of the C-terminal domain of the ε subunit (εCTD)thatblockscatalytic turnover. Thisregulation by ε is believed to play an important role in maintaining viability of the cell. Recent development in the field of antibiotics has validated ATP synthase as a drug target against pathogenic bacteria. Thus, there is a renewed interest in studying the role of the ε subunit in regulation of the enzyme and exploiting it to develop antimicrobials that can kill pathogenic bacteria. The present work describes advances in our understanding of the regulatory interactions of εCTD in E. coli ATP synthase.In the first approach, we used an optical binding assay to understand the transitions of εCTD between its active and inhibitory conformations.Using different ligands we revealedthe relationship between ADP inhibition and ε inhibition. In the second novel approach, the terminal five amino acids of εCTD were deleted to observe the effects on in vivo and in vitro functions of ATP synthase. The results obtained from these studies advance our understanding of εinhibition inbacteria and also provide a noveltarget within bacterial ATP synthase to obtain antibacterial drugs.
  • SHP-1-dependent macrophage responses mediate virus-induced myositis and demyelinating disease.

    Massa, Paul; Watson, Neva (2015)
    Regulation of inflammatory immune responses to pathogenic microbial infections is critical for protecting against extensive tissue damage and chronic inflammation. Correspondingly, genes associated with inflammatory immune responses have been identified as potential genetic risk factors for chronic inflammatory diseases including multiple sclerosis (MS) and idiopathic inflammatory myopathies (IIMs). This dissertation will focus on characterizing how the key immune regulator, Src-homology 2 containing protein tyrosine phosphatase-1 (SHP-1), controls virus-induced inflammatory diseases in the central nervous system (CNS) and skeletal muscle. We previously reported that SHP-1 inhibits proinflammatory macrophage-mediated CNS demyelinating disease during Theiler’s murine encephalomyelitis (TMEV) infection in mice. Presently, we describe that SHP-1 controls TMEV-induced dystrophic calcification of skeletal muscle. Muscle-infiltrating macrophages displayed a proinflammatory M1-like phenotype and promoted muscle calcification in WT mice, whereas an increased infiltration of macrophages with a reduced M1 signature corresponded with absence of muscle disease in SHP-1-/-mice. These studies reveal SHP-1 as a key regulatory gene mediating CNS and skeletal muscle disease in response to a virus trigger.Proinflammatory macrophages promoted tissue damage in either skeletal muscle of WT mice or CNS of SHP-1-deficient mice following TMEV infection. We thus attempted to determine if SHP-1 activity within macrophages was sufficient to control the outcome of TMEV infection using multiple genetic approaches. However, since these cells have a high turnover rate, and SHP-1 was strongly induced in macrophages by TMEV infection, these approaches were not sufficient to address whether SHP-1 activity specifically within macrophages mediates tissue-specific disease outcomes following TMEV infection. The studies described here suggest that SHP-1 affects macrophage maturation in peripheral (muscle) and immune-privileged (CNS) tissues in opposite ways. However, SHP-1 inhibited inflammatory monocyte CCR2 expression and subsequent infiltration into both of these major sites of infection, indicating that additional environmental cues mediated by SHP-1 are needed to drive tissue-specific maturation of pathogenic M1-like macrophages in either the CNS or muscle, to explain tissue-specific disease outcomes in SHP-1-deficient mice. Thus, this dissertation characterized unique mechanisms by which SHP-1 mediates inflammatory responses to virus infections, and has revealed SHP-1 and proinflammatory M1-like macrophages as essential mediators of myositis and demyelinating CNS disease.
  • REELIN SIGNALING COORDINATES DENDRITICINITIATION AND CELLULAR POSITIONING BY NEURITESTABILIZATION

    Olson, Eric; O’Dell, Ryan (2015)
    The laminar organization characteristic of the adult mammalian neocortex is a product of the precise coordination of neuronal proliferation, migration, and differentiation. Among these processes, the biological signals controlling apical dendrite initiation and targeting are not completely understood.The secreted ligand Reelin is a largeextracellular matrix glycoprotein localized to the axonal plexus of themarginal zone, and mutations areassociated with severe disruptions in cellular organization in laminated brain regions. Although the Reelin signaling pathway has been traditionally describedas a modulator of neuronal migration, recent evidence suggests Reelin controlsneuronal orientation and subsequent dendritogenesis into the overlying marginal zoneduring a period of early cortical development known as preplate splitting.To explicitly characterize how Reelin coordinates the transition between migration and dendritogenesis and controls polarized apical dendrite initiation and growth, an ex uteroexplant model of early cortical developmentwas used for fixed tissue and multiphoton live imaging analysis. Our investigations revealed the apical dendrite of cortical neurons emerges via direct transformation of the leading process during terminal translocation.Both throughoutand after this migratory phase, the dendriticarbor demonstrated significant increases in growth and branching, typically initiatedafter leading process entryinto the Reelin-rich marginal zone.In the absence of Reelin signaling, mutant cortices demonstrated a significant proportion of neurons that successfully translocated, but showed unstable arbors and marginal zone avoidance after migration arrest. Application of exogenous Reelin protein rescued dendritekinetics and polarity within4 hours, resultinginthe retraction of tangentially orienteddendritessimultaneous with the extension of a highly branched,apicallyoriented primary process. These findings suggesta precise role of Reelin signaling in early cortical development in proper neuronal polarization and stabledendrite outgrowth into the marginal zone, an area otherwiseexclusionary for dendrites. Furthermore, it is suggested that appropriate dendritic arbor elaborationinto the marginal zone may not only promote terminal translocation, but also definesthe final position of migration arrest.Thisbody of work thus offers an important advancement in understanding Reelin’s role in polarized dendritic outgrowth and the subsequent knock-on effectsassociated withperturbationsof this signaling pathway.
  • Metabolic Control of Autoimmunity in the Liver

    Perl, Andras; Oaks, Zachary (2016)
    Autophagy,literally meaning “self-eating,” is an integral part of cellularturnover of damaged organelles and proteins.This process is inextricably linked to mitochondrial function and turnover. Mitochondria can be degraded viaautophagy, known as mitophagy, as well as donate lipid membraneto generate autophagosomes fordigestingother organelles and proteins. On a larger scale, autophagy is essential for organ homeostasis. In the liver, autophagy ensures the turnover of damaged mitochondria that may otherwise increase oxidative stress which modifies DNA, proteins, and lipids resulting in the production of autoantigens or neoplasia. We investigated the role of autophagy and mitochondrial dysfunction prior to disease onset in mouse models of systemic lupus erythematosus (SLE). Patients and mice with SLE exhibit overexpression of transaldolase (TAL) and show predisposition to anti-phospholipid antibody production and associated liver diseases, including hepatocellular carcinoma. Wediscovered deficient mitophagy in the liver of lupus-prone mice prior to disease onset. Furthermore, these mice had increased mitochondrial respirationwith concomitant inner membrane hyperpolarization. These changes were coupled to overexpression of Rab4A, which depletes Drp1and thus inhibitsmitophagy.In addition,activation of complex I of the mechanistic target of rapamycin (mTORC1)was noted along with enhanced production of autoantibodies against mitochondrial phospholipids in lupus-prone mice. These changes were reversed by blockade of mTORC1 by rapamycin treatment in vivo. We then examined the role of TAL, a key enzyme of the pentose phosphate pathway (PPP) in mitochondrial dysfunction and oxidative stress. TAL-deficientmice showedincreased mitochondrial electron transport chain (ETC) activity and mTORC1 activation andreduced autophagy.Since inactivation of TAL caused oxidative stress via depletion of NADPH, we tested the hypothesis that aldose reductase(AR), a NADPH dependent enzymecan correct this metabolic defect without reversing the accumulation of TAL-specific substrates, sedoheptulose 7-phosphate and erythrose 4-phosphate. Moreover, deletion of AR reversed mTORC1 activation without affecting enhanced mitochondrial ETC activity or diminished autophagy. On a more global scale, predisposition to neoplasia and acetaminophen-induced liver failurewere reversed, while anti-phospholipid autoantibody production and liver fibrosis persisted in TAL/AR double-knockout mice indicatingthat the PPPmay act as a metabolic rheostat of organ-specific disease pathogenesis.
  • GENESIS AND MAINTENANCE OF LONG-TERM IgM+ T-BET+ B CELLS

    Papillion, Amber (2017)
    IgM memory cells are recognized as an important component of B cell memory, based on several studies both in mice and humans. Our studies of B cells elicited in response to ehrlichial infection identified a population of CD11c/T-bet-positive IgM memory cells and an IgM T-bet-positive bone marrow antibody-secreting cell population (ASCs). The origin of these populations was unknown, although an early T-independent spleen CD11c-and T-bet-positive IgM plasmablast population precedes both, suggesting a linear relationship. The majority of IgM memory cells detected after day 30 post-infection had undergone somatic hypermutation, indicating that they expressed activation-induced cytidine deaminase (AID). Therefore, to identify early AID-expressing precursor cells, we infected an AID-regulated tamoxifen-inducible Cre-recombinase-EYFP reporter strain. Tamoxifen administration led to labeling of both the IgM memory cells and bone marrow ASCs on day 30 and later post-infection. High frequencies of labeled cells were identified on day 30 post-infection,following tamoxifen administration on day 10 post-infection. Both IgM memory cells and IgM bone marrow ASCs were labeled when tamoxifen was administered as early as day 4 post-infection. We also identified mechanisms involved in maintenance of the IgM bone marrow ASCs and IgM+ memory cells, namely proliferation and FcγRIIb respectively. BrdU studies revealed that the bone marrow IgM ASCs were maintained by proliferation, unlike the IgM memory cells. RNAseq analysis revealed a 2-fold higher expression of inhibitory Fc receptor, FcγRIIb. Because FcγRIIb inhibits B cell activation, we hypothesized that FcγRIIb negatively regulates IgM+ memory B cells by binding immune complexes present during low-level chronic infection. E. murisinfection of FcγRIIb-deficientmice revealed a 3-fold expansion of the IgM+ memory 30 days post-infection. We further demonstrated that the expansion of the IgM+ memory cells was not due to increased proliferation, but a decrease of apoptosis, due to a lack of Fas expression in FcγRIIb-deficient mice. This result was mimicked in AID-deficient mice, which lack the ability to class switch to IgG and make immune complexes, revealing a role for immune complexes in regulating IgM+ memory. Altogether, these studies demonstrate a novel germinal center-independent pathway for the generation of two distinct long-term IgM-positive B cell populations.
  • Functional alterations and rhythmic disturbances by pan-histone deacetylase inhibition in the heart.

    Veenstra, Richard; Patel, Dakshesh (2016)
    Histone acetyl transferases (HATs) and histone deacetylases (HDACs) maintain a dynamic balance of acetylation and deacetylation of histone and non-histone proteins. HDAC inhibitors are small molecules anti-cancer therapeutics that exhibited dose limiting cardiac toxicities during clinical and preclinical trials. Multiple instances of abnormal T-waves, ST segment depression, QT prolongation, grade 3 sinus bradycardia and non- circumstantial deaths have been observed in patients. The underlying electrophysiological and molecular mechanism of these cardiac side-effects are poorly understood. In our in vivo ECG monitoring using Data Science International® telemetry transmitters, mice injected with panobinostat showed ventricular tachychardic and atrial fibrillation episodes with significant prolongation of ST, QT and QTc intervals. In whole cell patch clamp studies, we observed no significant change in transient and steady state K currents in myocyte ventricular cultures suggesting no role of hERG currents in ventricular arrhythmias. The majority (>90%) of congenital and drug induced QT prolongation is caused by alterations of hERG (IKr, Kv11.1) current. Interestingly, we observed significant reductions of INa and gap junctional conductance along with reductions in protein expression of Nav1.5 & Cx43 in vivo and in vitro. We conclude that pan-HDAC inhibition reduced cardiac INa density and gap junctional coupling with unaltered late INa and K+ currents explaining the cardiac abnormalities exhibited by panHDAC inhibitors. Decreased gap junctional coupling can enhance triggered activity by limiting electrotonic inhibition, combined with reduced INa density which can lead to slow myocardial conduction. Both of them taken together, increases the vulnerability to reentrant arrhythmias.
  • THE EFFECTS OF VARYING ACTIN AND CAPPING PROTEIN CONCENTRATIONS ON ACTIN PATCH DYNAMICS IN FISSION YEAST

    Sirotkin; Plante, Kyle (2015)
    Actin assembly into structures called endocytic actin patches is directly responsible for driving endocytic invagination and internalization. We tested the predictions made from the mathematical modeling of the Dendritic Nucleation Model of actin assembly at sites of clathrin-mediated endocytosis in fission yeast (Berro et al.; Sirotkin et al., 2010). The model predicts that increasing the concentration of cytoplasmic actin or deleting capping protein will cause an increase in the extent and the rate of actin assembly in actin patches. Conversely, the model predicts that increasing the concentration of capping protein or decreasing the actin concentration will cause a decrease in the extent and the rate of actin assembly in actin patches. To test these predictions, we used the actin cross-linking protein, fimbrin Fim1 tagged with a fluorescent protein to measure actin patch dynamics in strains that over- or under-express actin, over-express capping protein, or have deletions of the two capping protein genes. In contrast to model predictions, we found that manipulating capping protein concentrations did not have a significant effect on the extent of actin patch assembly and affected the rates of assembly to a lesser degree than expected from the model, suggesting that capping protein is not the only factor that limits actin patch assembly. Surprisingly, changes in the concentration of actin resulted in changes in the number of patches in a cell, suggesting that the concentration of actin is more important in controlling the initiation of new patches rather than in patch assembly. Through studying the biochemical pathway of actin assembly directly in living cells, we were able to gain insights into previously under-appreciated aspects of the mechanism of actin assembly at the sites of clathrin-mediated endocytosis.
  • THE TUMOR SUPPRESSIVE ROLE OF MONOGLYCERIDE LIPASE IN LUNG CANCER

    Huang, Ying; Liu, Renyan (2017)
    Monoglyceride lipase (MGL) is a serine hydrolase that hydrolyzes 2-monoglycerides and produces fatty acid and glycerol. Our previous studies showed that there was MGL deficiency in the majority of human lung, breast, and colorectal cancer tissues as compared with normal tissues. Further studies suggested that MGL was a potential tumor-suppressor in the development of colorectal cancer. However, paradoxical findings about the role of MGL in tumorigenesis have been reported. It is therefore important to further elucidate the function of MGL in tumorigenesis. To that end, we generated MGL-knockout mouse and found that MGL knockout led to tumor formation in multiple organs/tissues of mice. Particularly, the major findings were lung adenocarcinomas. In cultured cells, MGL deletion enhanced cell proliferation and induced cellular transformation. Further molecular studies demonstrated that MGL suppressed EGFR signaling, NF-κB activity, and COX-2 expression. Deficiency of MGL may lead to over-activation of EGFR, NF-κB, and COX-2, and therefore contribute to tumorigenesis. We also found that MGL over-expression induced remarkable cancer cell apoptosis, and increased cleavage of caspase-8, caspase-9, caspase-3 and PARP. MGLinduced apoptosis thus involves both the intrinsic mitochondria-mediated and extrinsic death receptor-initiated apoptosis pathways. Most importantly, our data indicated that MGL interacted with a potent inhibitor of apoptosis, XIAP, and significantly reduced XIAP protein stability, which may unleash caspase-9 and caspase-3 from XIAP inhibition and thereby promote apoptosis. Some additional findings about MGL showed that MGL localization to lipid droplets was important for its regulation of cell growth and pro-tumorigenic signaling while MGL’s lipase activity is dispensable for these effects. We identified Asp-115 (D115) of MGL as the most important amino acid residue in mediating MGL localization to lipid droplets. We also found that MGL deficiency promotes lipid droplet formation and cellular lipid accumulation, which potentially promotes cancer cell survival under stressful conditions. Overall, our in vitro and in vivo data strongly support the notion that MGL is a tumor suppressor. The molecular findings about MGL may have revealed certain targets for personalized cancer therapy in the context of MGL deficiency and they also implicate potential roles of MGL in inflammation, immunity, and metabolism.

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