Now showing items 21-40 of 103

    • Mechanism of gene regulation of HNF4α

      Lu, Hong; Guo, Shangdong (2018)
      Hepatocyte Nuclear Factor 4 alpha(HNF4α)is a masterregulatorthat modulatesthe liver development andfunction. The dysfunction of HNF4αcauses multiplehumandiseases, such as hepatocellular carcinoma(HCC) and maturity onset diabetes of the young1 (MODY1). Incontrast,the restoration of HNF4α can inhibit the development of liver cancer and improve the liver function simultaneously.However, HNF4αis anorphan nuclear receptor whose activating ligand remains elusive. Therefore, an alternative approach to enhance the HNF4αactivity is to up-regulate the proteinexpression.While a great progress has been made on the functional study of HNF4α,the mechanistic details regarding the gene regulationofHNF4αare still a vast knowledge gap. The present study was aimed to investigatethe mechanism of gene regulation ofHNF4αsystematically. In chapter 2 and chapter 3, we reported the strong translational inhibition of both humanand mouse HNF4αinduced by the nucleicacid secondary structuretermed “G-quadruplex”(G4)within the 5` untranslated region(UTR).By performing the deletion/mutation studies, we determined the compositionof the G4in HNF4A 5`UTR. We further speculated thatthis G4 required the adjacent cis-elements, such as the RNA-binding proteins and other secondary structures, to form a conjunction for the strong translational inhibition. We for the first time reported the RNA-G4 induced translational repression within the 5`UTR of a tumor suppressor and highlighted the significanceof the “biostability”of G4s in exerting their biological functions. In chapter 4, we conductedacomprehensivestudy that coveredthe auto-regulation, transcriptionalregulation and transactivation activity of HNF4α. By creatingvariousreporter constructs, we were able to validate the self-stimulation of HNF4αand discovered the strong correlation between HNF4αand its corresponding anti-sense RNA, HNF4A-AS1. Additionally, we identified novel HNF4αmutations such as Q277X that may affect the crosstalk of HNF4αwith other transcriptionfactors.Overall, the novel findings from our study shedlight on the gene regulation of HNF4αand providefurther insights into ourultimate goal that is to up-regulate HNF4αprotein expression/activity to treat human diseases.
    • Reelin Signaling in Oligodendrocyte Progenitor Cell Migration

      Osterhout, Donna; BHATTI, HARNEET (2016)
      Oligodendroglial progenitor cells (OPCs) are the precursors to the myelinating oligodendrocytes in the central nervous system (CNS). These cells are produced in the ventral neuroepithelium at later stages of cortical development, migrating into the cortex where they contact axons and differentiate, ultimately forming a myelin membrane. During the process of differentiation, OPCs undergo significant morphological changes, extending many processes which will make contact with axons. Once in contact with an axon, the oligodendrocyte process expands and begins to form the myelin membrane which will ensheathe the axon. Reelin is a highly conserved secretory glycoprotein, which has acritical role in directing neuronal migration. Reelin orchestrates the proper cortical layer formation and neuronal organization during brain development. In the absence of Reelin, the cerebral crotex is disorganized, with inverted cortical layers, generating devastating biological effects. Reelin acts through several cellular receptors, activating numerous downstream effectors and complex signaling cascades. If elements of the Reelin signalling pathway are disrupted,similar defects in migration can occur.Oligodendroglial cells, from the early progenitor cells to the mature myelinating cells secrete Reelin, but also express a receptor for Reelin and criticalelements of the intracellular Reelin signaling pathways. It is not known if these cells canrespond to Reelin. In this thesis, we examined the effects of Reelin on oligodendroglial cells, using both in vitroand in vivomethods. We demostrate a potentialrole for Reelin in modulating oligodendrocyte migration, but also identify a novel aspect ofReelin signalling in the biology of oligodendroglia.
    • THE ROLE OFNORMAL AND ONCOGENICJANUS KINASE 2 IN HEMATOPOIETIC STEM CELLS

      Mohi, Golam; AKADA, HAJIME (2014)
      During my Ph.D. training, I first experimentally proved that the expression of oncogenic Jak2V617F wassufficient to induce MPNs and transformed only HSCs into CSCs for developing MPNs. Thus, it is criticalto understand the role of both normal and oncogenic Jak2 in HSCs to find the mechanism to cureJak2V617F-positive MPNs. Therefore, I have mainly studied two major questions in Jak2:1) The role of normal Jak2 in hematopoietic stem cells for adult hematopoiesis2) The role of oncogenic form of Jak2, Jak2V617F, in cancer stem cells for MPN developmentFirst question has not been addressed since 1998, because conventional Jak2 knock-out mice wereembryonic lethal. Thus, I hypothesized that Jak2 plays a pivotal role in adult hematopoietic stem cellmaintenance. I successfully prove that Jak2 is the one of key regulators of HSCs. Conditional Jak2 deletionin mice caused an irreversible HSCs impairment. My data strongly suggest that Jak2 plays a critical role inthe maintenance of quiescence, survival and self-renewal of adult HSCs.Second question has been studied after the discovery of a somatic point mutation, Jak2V617F, in a majorityof patients with MPNs in 2005. I hypothesized that this oncogenic mutation confers unique properties inCSCs maintenance for MPNs development. Surprisingly, I found that the site of leukemogenesis shiftedfrom BM to spleen, and spleen became the major source of CSCs for Jak2V617F-positive MPNs. The age-associated progressive expansion of CSCs was seen in spleen. Splenic-CSCs were capable to propagateMPN disease and possessed a greater proliferative advantage than BM-CSCs. The Jak2V617F-CSCsestablished a positive-feedback mechanism with CD169+ macrophage progenitors. Depletion of CD169+macrophage progenitors reduced the number of Jak2V617F-CSCs. Gene profiling revealed that splenic-CSCs have distinct gene expression compared to BM-CSCs. Together, I demonstrated that Jak2V617F-CSCs are maintained in spleen for long-term MPN progression.By utilizing gene analysis data from two projects, I discovered a set of unique genes/pathways regulated byonly Jak2V617F but not by wildtype Jak2. All together, my Ph.D. researches provided the potential genetarget a novel therapy for Jak2V617F-positive MPNs.
    • STRUCTURE AND FORMATION OF PERINEURONAL NETS: A UNIQUE FORM OF EXTRACELLULAR MATRIX IN THE CENTRAL NERVOUS SYSTEM

      Russell Matthews; Sinha, Ashis (2021)
      The complex organization and trajectory of development of the central nervous system (CNS) depends not only on cell-cell interactions but also on interactions of cells with the extracellular environment. The extracellular environment in the CNS contains an organized network of proteins, glycans and glycoproteins and comprises what is called the CNS extracellular matrix (ECM). Components of this ECM are dynamically regulated not just during development but are also involved in various neurobiological processes in the adult. They are implicated in normal nervous system physiology as well as in nervous system disease and pathology. A complete understanding of the CNS therefore, requires an understanding of the composition, structure and function of the neural ECM also. Our laboratory focuses on a specialized substructure of the neural ECM, called perineuronal nets (PNNs). PNNs are conspicuous structures within the neural ECM and are thought to be key regulators of neuronal plasticity. Traditionally their role has been demonstrated in regulating forms of plasticity seen during development, but recent work has implicated PNNs in other forms of learning and plasticity across various brain regions as well. Interest in these structures has been further peaked by studies linking their alterations to a variety of neurological and neuropsychiatric diseases such as Alzheimer's disease and schizophrenia. However, despite this growing interest in PNNs, the mechanisms by which they modulate neural functions are poorly understood. The limited mechanistic understanding of PNN function is derived primarily from the fact that there are no existing models, tools or techniques that specifically target them without also disrupting the surrounding neural ECM. Therefore, a rigorous investigation of PNN function to date has not been possible. Our inability to specifically target PNNs is driven by an incomplete of understanding of their molecular composition and structure. While PNNs comprise of many known ECM components which are broadly expressed in the CNS ECM, PNNs appear clearly distinct from their surroundings. They are highly ordered and stable and show a regular organization and geometry not present in the diffuse neural ECM. The exact molecular mechanisms by which various PNN components come together and aggregate on the cell surface to form these structures however, is not iii clearly known. The primary goal of our laboratory has therefore been to determine the molecular structure, composition and formation of PNNs. Through our work described in chapters 2 and 3 of this thesis, we were able to develop a new model of PNN structure. In our proposed model, PNN components are bound to the cell surface by two distinct types of interactions, one dependent on the classical HA scaffold of PNNs and the other mediated by a complex formed by the ECM glycoprotein tenascin-R (TNR) and the chondroitin sulfate proteoglycan receptor protein tyrosine phosphatase zeta (RPTPz). Our work also allowed us to provide evidence that PNN components are immobilized on the neuronal surface by a GPI-linked mechanism. We identify the GPI-linked protein contactin-1 (CNTN1) as a key receptor molecule for PNNs. To our knowledge, this is the first ever identified receptor for PNNs. We feel that our findings presented here give us important insight into PNN structure and represent the initial important steps in understanding the formation of this unique ECM subcompartment.
    • T Cell Factor-1 (TCF-1) Regulates Mature Alloactivated T Cells to Separate GVHD From GVL

      Mobin Karimi; Harris, Rebecca (2021)
      Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment used for patients with cancer or other hematological malignancies. However, widespread use of this treatment is hindered by development of graft-versus-host disease (GVHD), a life-threatening complication of allo-HSCT. Mature donor T cells in the graft mediate GVHD, but also help kill residual malignant cells in the patient by the graft-versus-leukemia (GVL) effect. Depletion of mature T cells from the graft eliminates this beneficial anti-tumor response. Mature T cells are also needed for proper stem cell engraftment. Therefore, current work has focused on how to modulate T cell signaling and function to separate GVHD from GVL. T Cell Factor-1 (TCF-1) is a T cell developmental transcription factor that is also important in some contexts for T cell activation. The role of TCF-1 in alloactivated mature T cells is completely unknown. To examine the role of TCF-1 in this context, a mouse model of allo-HSCT leading to GVHD/GVL was used to study T cells from mice with a T cell-specific deletion of TCF-1. This work showed that loss of TCF-1 separates GVHD from GVL, with reduced disease severity and persistence yet maintained GVL effects. TCF-1 affects alloactivated T cell phenotypes and suppressive profiles, as well as the major T cell functions (proliferation, migration, and cytokine/cytotoxic mediator production). TCF-1 also controls alloactivated T cell survival, apoptosis, and gene expression programs. The regulation of these functions and programs by TCF-1 is distinct in CD4 versus CD8 T cells. TCF-1 also controls two unique T cell subsets - stem-like CXCR5+ CD8+ T cells, and CD25- noncanonical Tregs. Therefore, TCF-1, or these two unique T cell types, may be a therapeutic target to improve allo-HSCT outcomes by separating GVHD from GVL effects. Expansion of CD25- Tregs during TCF-1 deficiency may also be useful for treatment of other T cell-mediated disorders as well.
    • Defining the Determinants of the Bok-IP3R Interaction and the Bok Interactome

      Richard Wojcikiewicz; Szczesniak, Laura (2021)
      Bok is a Bcl-2 protein family member that is often grouped with the pro-apoptotic family members Bax and Bak due to high sequence homology and because exogenously overexpressed Bok induces apoptosis by causing mitochondrial outer membrane permeabilization. However, the cellular roles of Bok remain unclear, as Bok KO cell lines and mice have failed to demonstrate a significant phenotype under normal conditions. Our lab discovered that Bok interacts with inositol 1,4,5-trisphosphate receptors (IP3Rs), tetrameric Ca2+ channels found in the ER membrane of mammalian cells that play an integral role in cell signaling. While other Bcl-2 family members have been reported to weakly interact with IP3Rs, the Bok-IP3R interaction is much more efficient, with essentially all cellular Bok constitutively bound to IP3Rs. We have generated full-length IP3R1 mutants that resolve the Bok-binding region to a small, unstructured loop in the cytosolic region of IP3R1 between _ helices 72 and 73. Additional bioinformatic analysis has revealed that the Bok-IP3R interaction is likely dependent upon helical and dynamic determinants within this loop. Interestingly, Bok KO cell lines demonstrate mitochondrial fragmentation and only minor changes in mitochondrial bioenergetics. We have investigated the role of Bok in mitochondrial dynamics through an in vivo proximity labeling technique known as TurboID. A TurboID-Bok fusion protein expressed in Bok KO HeLas has identified several mitochondrial fission proteins through mass spectroscopy analysis, suggesting that Bok acts at mitochondria-ER contact sites to inhibit fission, and this function may be independent of the Bok-IP3R interaction. The results provided from binding studies and proximity labeling have furthered our knowledge of the Bok-IP3R interaction and of Bok itself to better define, or re-define, the role of Bok within the cell. Understanding how and why these interactions occur will help us further understand fundamental cellular processes in health and human disease.
    • Identification of TIMP2 as the first secretory co-chaperone of eHSP90

      Dimitra Bourboulia; Baker-Williams, Alexander J. (2021)
      Heat Shock Protein- 90 (HSP90) is an essential molecular chaperone. HSP90 relies on its intrinsic ATPase activity as well as interactions with co-chaperone proteins to chaperone its clients. HSP90 is also an extracellular protein, performing both a signaling and chaperoning role. Extracellular client, matrix metalloproteinase-2 (MMP2) relies on HSP90 for its stability. MMP2 mediates extracellular matrix remodeling through its gelatinolytic activity. MMP2 activity is also tightly regulated by its endogenous inhibitor, the Tissue Inhibitor of Metalloproteinase-2 (TIMP2). At present how HSP90 performs its chaperoning role in the extracellular matrix is uncertain. In this thesis, I describe that TIMP2 acts as the first bona fide extracellular co-chaperone of eHSP90, and show that TIMP2 is a stress inducible protein. I describe how TIMP2 directly interacts with HSP90, and how TIMP2 decelerates the HSP90 ATPase cycle. TIMP2 also sensitizes HSP90 to both ATP and N-terminal pharmaceuticals. Overall, TIMP2 acts as both a scaffold and a disruptor of the client/chaperone relationship between MMP2 and HSP90, performing both a HSP90 co-chaperone and MMP2 inhibitor role, non-mutually exclusively. The activatory co-chaperone AHA1 competes with TIMP2 for HSP90 binding. TIMP2 and AHA1 are able to form two independent ternary complexes with MMP2 and HSP90; as a result, the TIMP2 complex is MMP2 proteolytically inactive and the AHA1, active. This competition is further described in vivo where it can be inhibited by both _AHA1 antibodies and TIMP2 AHA1 antibodies and TIMP2 exogenous protein treatments, whilst induced following AHA1 protein and _AHA1 antibodies and TIMP2 TIMP2 antibody treatments. Finally, the role of phos-Y-TIMP2 was examined in relation to its interaction with HSP90. To address this, a novel methodology to purify hTIMP2 from E.Coli without previously necessary refolding strategies in a scale-able manner suitable for therapeutic TIMP2 treatments, was developed. Wild type recombinant human TIMP2 and phospho-mutants Y90E, Y90F, and TE (Y62E, Y90E, Y165E) were purified, were inhibitory towards MMP2, and modulated TIMP2 interaction with HSP90. Taken together, I demonstrate how extracellular HSP90 is regulated by co-chaperones to facilitate the chaperoning of pro-invasive client, MMP2. It further shows ways in which we can manipulate this system to promote an inactive MMP2 protease, a key strategy in cancer therapeutics.
    • Memory and Effector B cell Responses to Viral and Intracellular Bacterial Infection

      Gary M. Winslow; Newell, Krista (2021)
      Infection with an intracellular pathogen presents the host with the immunological challenge of intracellular access and of clearing infection without excessive damage to host tissues. This challenge was long thought to be addressed primarily by cell-mediated immunity, but is now known to include a significant humoral component. To better understand the B cell-mediated contribution to intracellular pathogen control, we investigated memory and effector B cell responses to the intracellular bacterial pathogen Ehrlichia muris, and SARS-CoV-2 infection. B cells expressing the transcription factor T-bet were identified in both mice and humans, and T-bet played an important role in directing antibody class switch recombination, but not in the generation of memory B cells during E. muris infection. T-bet expression was identified in cells resembling murine B-1 B cells, an innate-like subset of B cells important for early T cell-independent B cell responses. These results suggest that T-bet expression in B-1 B cells may contribute to their participation in the early B cell response to murine intracellular bacterial infection. Following human SARS-CoV-2 infection, we revealed that in addition to the canonical class-switched B cell memory response, the presence of a substantial pool of peripheral blood unswitched IgM+ memory B cells was correlated with reduced symptom duration and enhanced generation of antigen-specific antibody. These IgM+ memory B cells were stable, unlike the contracting plasmablast response. These studies underscore the importance of innate and unswitched B cell subsets to the functional plasticity of the humoral response and contribute to our understanding of correlates of innate protection and adaptive immunity.
    • POLY-OMIC PROFILES OF SCHIZOPHRENIA AND BIPOLAR DISORDER

      Glatt, Stephen J.; Hess, Jonathan (2017)
      In1899, psychiatrist Emil Krapaelin introduced a separation between schizophrenia (SZ) and bipolar disorder (BD), formerly “dementia praecox” and“manic-depressive disorder”, which came to be known as the Krapaelinian dichotomy, and has prevailed over the past century (Kraepelin, 1904). Although Emil Krapaelin postulated that these are distinct entities, multiple converging lines of evidence suggest that SZ and BD have a shared etiology: (1) first-degree relatives of a SZ-affected individual are at higher risk for BD than the general population, and vice versa (Lichtenstein et al., 2009), (2) recent work from genome-wide association studies (GWAS) and rare variant studies revealed that SZ and BD share common risk genes, suggesting that these disorders share a set of molecular substrates, and (3) second-generation antipsychotics exhibit effectiveness in ameliorating psychosis and mania (Buckley, 2008). SZ and BD are highly heritable mental illnesses with a lifetime prevalence near 1%. Onset typically occurs in late adolescence to early adulthood. Their etiology is complex and multi-factorial. SZ and BD are among the leading causes of disability around the globe(Global Burden of Disease Study 2013 Collaborators, 2015). There isa constellation of symptoms related toSZ, including hallucinations (e.g., auditory, olfactory, visual), delusions (e.g., persecutions, grandiosity), thought disturbances, affective flattening, and anhedonia. SZ and BD have clinical resemblances like psychosis, though this is more widely recognized as a hallmark of SZ. The core feature of BD is extreme changes in mood ranging from periods of mania followed by severe depression, which is also referred to as “switching”. Drugs for treating SZ and BD have changed very little over the past 50 years, and those that are used today are not always effective and can elicit severe side effects. SZ and BD research is evolving rapidly but our understanding of these disorders is still in its infancy. One of the major advances in the field has been the “big data” revolution. Technological advances have been a critical driving force of this revolution, including emergence of DNA microarray chips for high-throughput genome-wide genotyping and gene expression profiling. These technologies became widely adopted in psychiatry and led to a proliferation of genome-and transcriptome-wide studies in psychiatry to aid in the discovery of novel genes and pathways related to mental illness. Despite SZ and BD having a strong genetic basis, identifying susceptibility genes was a significant challenge. Combining data across laboratories became a fundamental strategy to overcome inherent weaknesses with statistical power and methodological biases, which has proven be to a fruitful strategy for GWAS (Cross Disorder Group of the Psychiatric Genomics Consortium, 2013; Ripke et al., 2014; Ruderfer et al., 2013; Sklar et al., 2011). Yet, a robust methodological and statistical framework for analyzing combined collections of gene expression data has been lacking in psychiatry. Microarray studies of SZ and BD suffered from low statistical power and drawbacks that affected their reproducibility (Draghici, Khatri, Eklund, & Szallasi, 2006; Evans, Watson, & Akil, 2003). Combining gene expression data from numerous sources and addressing methodological issues may help to uncover reliable molecular associations. Even though the relevance of gene expression to physiology is not always clear, gene expression abnormalities in mental illness can provide fundamental insight into gene regulatory networks in brain and peripheral tissues, and provide a framework for interpreting genomics data. Integrating findings between GWAS and gene expression studies has the potential to elucidate the etiological overlap of SZ and BD. Moreover, gene expression signatures of mental illnesses may have biomarker utility and set up a foundation for identifying better drug targets. Data sharing is now a common place. Although microarrays are gradually being replaced by more sensitive and precise technologies such as next-generation sequencing, data harmonizing will be a pervasive issue unless dealt with now.In this dissertation, I present two review papers describing the current state of SZ and BD genetics research followed by three primary research studies that I performed to answer these prevailing questions: (1) what are the genes, pathways, and regulatory elements that relate to risk for SZ and BD, and are these similar or different across disorders? (2) what genes and pathways are abnormally expressed in SZ and BD, and might these differences converge with genomic evidence? (3) can differences between SZ and BD reflected in gene expression profiles offer biomarker utility and a basis for developing disorder-specific classifiers?My primary hypothesis for this work is SZ and BD exhibit overlapping abnormalities across pathways related to neurodevelopment, neurotransmission, and immunity/cellular response to stressors; furthermore, these abnormalities are relevant for pathophysiology. My dissertation work encapsulates the development of methodologies and computational tools to analyze large “poly-omics” data sets, i.e., jointly analyzing genomic, epigenomic, and transcriptomic data to identify abnormalities gene expression regulation and molecular substrates that are common between and unique to SZ and BD. My work uncovered convergent evidence of dysregulation among genes, pathways, and regulatory molecules associated with SZ and BD. Major outcomes of this thesis may help to lay the groundwork for causal inference of the effect of genetic variants on cellular phenotypes, biological sub-typing of mental illness through gene expression profiling, and rational drug design.
    • DECIPHERINGGLUTAMATERGIC NEUROTRANSMITTER SPECIFICATION IN THE ZEBRAFISH SPINAL CORD

      Lewis, Kate; Hilinski, William (2016)
      The correct specification of neurotransmitter phenotypes is crucial for properly functioning neuronal circuitry. Neurons specify their neurotransmitter phenotypes via transcription factors that they express as they differentiate. Often, transcription factors that specify neurotransmitter phenotypes are expressed in multiple populations with the same neurotransmitter phenotype. Since, the transcription factors that specify this characteristic are not yet known for all spinal cord glutamatergic populations, we identified additional transcription factors expressed at relatively higher levels in glutamatergic neurons compared to inhibitory neurons. We have functionally tested three of these (Lmx1bb, Skor1a and Skor1b) to determine if they are required for correct spinal, glutamatergic phenotypes. We demonstrate that Lmx1b likely maintains a subset of glutamatergicphenotypes in the spinal cord. In lmx1bb mutant embryos, the number of cells that initially express glutamatergic markers are unchanged but become reduced at 36 h and to a greater degree at 48 h. In contrast, we observe no changes in the total number of dI5 or V0v neurons, which express lmx1bb,nor do we detect elevated levels of apoptosis between 36 h and 48 h in lmx1bb mutants. Lastly, we show that at least some of the cells that lose their glutamatergic neurotransmitter phenotype are likely to be V0v cells.Additionally, we demonstrate that skor1a and skor1b are expressed predominantly by glutamatergic spinal interneurons, many of which are V0v neurons. When skor1a and skor1b are knocked-down, we observe a significant reduction in the number of glutamatergic neurons and no change in the number of inhibitory neurons suggesting that these genesmay be required to specify the glutamatergic neurotransmitter phenotype of a subset of spinal neurons. In parallel studies, it was shown that evx1 and evx2, genes expressed exclusively by V0v spinal neurons, are required to specify the V0v glutamatergic phenotype. Interestingly, we show that lmx1bb, skor1a and skor1b require evx1 and evx2 for their expression. In summary, these results suggest that skor1, skor1b and lmx1bb may function downstream of evx1 and evx2 to specify and/ormaintain the glutamatergic neurotransmitter phenotype ofat least a subset of V0v neurons.
    • Rational Design of Protein-Based Biosensors Using Engineered Binding-Induced Conformational Switches

      Loh, Stewart; ZHENG, HUIMEI (2014)
      Biosensor development continues to be driven by the growing need to accurately detect and monitor analytes with many biotechnology, clinical, agriculture, and military applications. With their well-established capacity for molecular recognition, proteins are the go-to choice of binding elements in many conventional sensor designs. Switchable proteins offer the potential of integrating analyte binding and signal transduction within a single molecule, thus reducing the need for complex and expensive detection equipment and opening the door to miniaturization and in vivo applications. The principal challenge is that the majority of natural binding proteins do not undergo a large-scale change in conformation upon target binding. This work describes two complementary protein design strategies for the rational conversion of ordinary binding proteins into ligand induced conformational switches for biosensing purposes. In the first approach, we applied the Alternate Frame Folding (AFF) mechanism to the human sulfiredoxin (hSrx) and the fibronectin (FN3) monobody scaffold towards the creation of an ATP biosensor and a customizable biosensor platform, respectively. In a second novel approach, the Protein Fragment Exchange (FREX) mechanism was demonstrated in a proof-of principle study that converts the FN3 scaffold into a biosensor, capable of genetic encoding and application in mammalian cells. While these designs were based on well established principles of protein folding and thermodynamics, the results obtained from these studies also offer important insights regarding protein sequence-structure-function relationships.
    • TIMP-2 PHOSPHORYLATION BY EXTRACELLULAR c-SRC REGULATES proMMP-2 ACTIVATION

      Bourboulia, Dimitra; Sánchez Pozo, Javier (2018)
      Matrix metalloproteinases (MMPs) are secreted zinc-dependent endopeptidases that are involved in many extracellular biological processes due to their matrix-degrading function. The majority of theseenzymes are released intothe extracellular space in their inactive form and require activation. The tissue inhibitors of metalloproteinases (TIMPs) are also secreted proteins and mainly function to inhibit all members of the MMP family. Interestingly, TIMP-2 also participates in the activationprocessof proMMP-2. Although the interaction between TIMP-2 and proMMP-2 has been known for decades, the molecular signal that triggersthis association has only recently beendetermined. Studies in our lab haveshown that TIMP-2 is tyrosine phosphorylatedby the c-Srctyrosinekinase. Also, phosphorylation of TIMP-2 Tyr90is essential for its interaction with proMMP-2in vivo. Our hypothesis is that c-Src-mediated TIMP-2 phosphorylation happens outside the cell. Here, wedemonstratethat TIMP-2 and c-Src are secreted through different secretory pathways and that TIMP-2 phosphorylation takes place in the extracellular space. Our workalso showsthatextracellularc-Srcisactive, reinforcing the fact that phosphorylation can happen extracellularly. We also hypothesize that extracellular c-Src plays a critical role in facilitating TIMP-2:proMMP-2 interaction. We first confirmed thatTIMP-2 and proMMP-2 endogenously interact only in cells containing endogenous c-Src. This interaction,as well as TIMP-2 phosphorylation,was blocked by treating cells with acustom-made anti-c-Src polyclonal antibody (pAb)that targets amino acids 84-110. We also showthat ananti-c-Src antibody that targets the first 79 amino acids does not inhibit TIMP-2 phosphorylation and interaction with proMMP-2. Therefore, since TIMP-2:proMMP-2 complex formation promotes proMMP-2 activation, we hypothesize that c-Src is an essential player in this process. Our data showsthatthe non-phosphorylatable TIMP-2Tyr90mutant does not promote proMMP-2 activation. Furthermore, pretreatment with the anti-c-Src pAbblockedTIMP-2-mediated proMMP-2 activation, whereasthe anti-c-Src mAb6 did not affect proMMP-2 activation. Overall, these findings provide further evidence that secreted c-Src-mediated TIMP-2 phosphorylation occurs in the extracellular space, where thesecretedkinase is also active. Moreover, c-Src is essential for TIMP-2:proMMP-2 complex formation as well as proMMP-2 activation.
    • Role of the SMC5/6 complex in DNA replication and DNA damage repair

      Feng, Wenyi; Peng, Jie (2017)
      The structural maintenance of chromosome (SMC) proteins form the core of Cohesin, Condensin and the Smc5/6 complex, which are essential for organization and metabolism of chromosomes during the cell cycle. The Smc5/6 complex is implicated a role in non-essential homologous recombination-mediated (HR) DNA repair. Inactivating Smc5/6 (via temperature-sensitivity mutant) in the S phase, but not in the G2 phase, causes mitotic failure. Hence, we hypothesize that the Smc5/6 complex has an major role in DNA replication. We analyzed the genome-wide DNA replication temporal program in different genetic backgrounds. Mapping replication fork-associated ssDNA in WT and rad53 cells in the presence of replication stress allowed us to separate early and late replication origins. Using this method, we revealed a strain background difference of origin usage in A364a and W303. We then studied the genome-wide replication dynamics in a hypomorphic mutant, smc6-P4, using a density transfer coupled with microarray method. Overall replication dynamics of the mutant are similar to that of the WT cells with exceptions in the early S phase. However, we captured a difference in the replication profile of an early S phase sample in the mutant, prompting the hypothesis that the mutant incorporates ribonucleotides and/or accumulates single-stranded DNA gaps during replication. We then tested whether we can exacerbate the DNA replication stress hypersensitivity of the smc6 mutant by inhibiting ribonucleotides excision repair (RER) pathway. Contrary to our expectation, impairment of ribonucleotide excision repair, as well as virtually all other DNA repair pathways, alleviated smc6 mutant’s hypersensitivity to induced replication stress. We propose that nucleotide incision with impaired Smc5/6 complex has a more disastrous outcome than the damage itself. We found that the smc6 mutant has a significant increase in ssDNA level under replication stress but inhibiting RER pathway decreases it. This result suggests that Smc5/6 complex prevents unsupervised ssDNA formation during DNA replication as a result of damage incision repair. We reason that excess ssDNA in the Smc5/6 defective cells may subsequently engage in toxic recombination. Our study thus provides novel perspectives for the role of the Smc5/ 6 complex during DNA replication.
    • EFFECTS OF FOCAL SEGMENTAL GLOMERULOSCLEROSIS-ASSOCIATED MUTATIONS ON MYOSIN 1E LOCALIZATION AND ACTIVITY

      Krendel, Mira; Karchin, Jing Bi (2015)
      Our lab has discovered that an actin-dependent molecular motor called Myosin 1e (Myo1e) is required for maintaining normal morphology and function in vivo of podocytes, a specialized epithelial cell in the kidney. We have found that Myo1e-null mice develop proteinuria, and mutations in the MYO1E gene, including missense mutations A159P and T119I, and nonsense mutation Y695X, have been identified in focal segmental glomerulosclerosis (FSGS), a primary kidney disease that often leads to end stage renal disease (ESRD). Based on these findings, we have proposed that Myo1e and especially its motor domain, plays a key role in regulating actin cytoskeleton organization in kidney podocytes. To study Myo1e activity at the junctions, we have used cell culture systems. We confirmed that Myo1e is a component of the podocyte slit diaphragm using glomerular fractionation assay and immune-gold labeling electron microscopy. Disruption of Myo1e motor activity by point mutation (A159P) completely disrupted Myo1e cellular localization and led to defective actin assembly at nascent cell-cell contacts. Domain mapping experiments in MDCK cells have suggested that the Myo1e TH2 domain is necessary, but not sufficient for its localization, but addition of the TH1 domain restores its localization to junctions. We have also found that the Myo1e SH3 domain interacts with ZO-1, a slit diaphragm and tight junction protein, in invitro pulldown assays, which might contribute to ZO-1 exchange activity at the junctions. Another FSGS-associated Myo1e motor domain mutation (T119I) also caused mis-localization of Myo1e in the cultured mouse podocytes, suggesting loss-of-function of the motor domain mutants. We have also shown that ZO-1 is not recruited to the nascent cell-cell contacts at the same time with the Myo1e T119I mutants. Finally, by using fission yeast as a model system, we have demonstrated that human kidney disease-associated mutations in fission yeast caused defects in yeast growth and endocytosis processes. Interestingly, after analyzing the colocalization patterns between the FSGS-associated Myo1 mutants and Chaperone Rng3, we have proposed that these two kidney disease-associated mutants likely possess different disease-causing mechanisms.Above all, we have concluded that Myo1e motor domain plays an important role in its localization and activity in podocyte actin cytoskeleton, which might be the link to the disease mechanism of FSGS at the molecular level.
    • STRUCTURAL BASIS OF THE FUNCTIONAL ROLES OF HUMAN AROMATASE

      Ghosh, Debashis; Lo, Jessica (2015)
      Humanaromatase(AROM) catalyzes theconversion of androgens to estrogensand is a major breast cancer drug target. Structural investigation has provided insights intothe active siteandaromatization mechanism.Utilization of the structural data has permitted rational design of a series of novel steroidal inhibitors. Investigation ofthe roles of key amino acids is facilitated by a recombinant AROM identical in crystal structureto the placental AROM.We use mutagenesis, chromatography, ultracentrifugation, spectrophotometry, enzyme kinetics, and X-ray crystallography to probe the roles of critical residues and the molecular basis of oligomerization. Furthermore, weevaluate the potencies of novel inhibitors and determine the structural basis of inhibition andselectivity. A critical active site residue D309 withan elevated pKa remainsprotonated at neutral pHand facilitatessubstrate binding and catalysis.The “gatekeeper” R192, linked to D309 via a watermolecule, is postulated to have a role in proton relay and substrate selectivity. D309N and R192Q mutants are virtually inactive supporting thehypothesis that both play keyrolesin aromatization.AROM oligomerization is driven bytheD-E loop of one moleculeand heme-proximal region of another via hydrogen bonding, electrostatic interactions between E181 and K440, and shape complementarity.Del7, generated by deletionof 7 residues in the D-E loop, experiences 65% reductionin activitydue to the loss of oligomer formation. Mutants Del4, E181A, and E181K exhibit normal enzymatic activity,and maintain some oligomeric interactions. The heme-proximal interface is also the putative coupling site of the reductasethatsupplieselectronsfor aromatization. The siteis larger than the active site, and at least twice aslarge asother P450s.MutantsK440Qand Y361Fof this region are virtuallyinactive.Collectivelythe results suggestfunctional significanceof oligomerization. Several newly designedAIs are superiortoexemestane, the steroidal AI currently used as a drug, in inhibition and anti-proliferation assays. The C6β-(pent-2-yn-1-yloxy) side chains ofthe most potent compoundspenetrate the access channelunique to AROM and havethe sameconformation asin the enzyme-free state.Astructural-based approachcan improve drug efficacy by improving specificity and selectivity, and reducing sideeffects.
    • ROLES FOR NETRIN-4 AND LAMININS CONTAINING THE β2 OR γ3 SUBUNITS IN CORNEAL DEVELOPMENT AND WOUND HEALING

      Brunken, William; Martino, Jeremiah (2016)
      Corneal diseases are among the top preventable causes of vision loss in the developed world. Injuries, infections, age-related diseases and congenital diseases are typically accompanied by defects in the extracellular matrix (ECM) or its remodeling. Laminins and laminin-related molecules, netrins, are ECM proteins that play an important yet not fully characterized role in corneal development, disease and wound recovery. With regulatory roles in cell proliferation, migration and neural guidance, the main research question addressed here is how netrin-4 and specific laminin subunits, i.e. the laminin β2 and γ3 chains, are involved in corneal development and wound recovery. A reverse genetics approach was employed to characterize the corneal phenotype in mice lacking the Ntn4, Lamb2, or Lamc3genes during development and wound healing.Immunohistochemistry data showed that netrin-4 and the laminin β2 and γ3 chains are components of corneal basement membranes. Genetic ablation of Lamb2, Lamc3or both genes led to hypo-proliferation of epithelial progenitors and hypo-innervation of the corneal surface, indicating regulatory roles in corneal development and possibly wound recovery. In addition to these effects at the surface, in the deep cornea, Descemet’s Membrane (DM) was thinner and endothelial cells displayed disruptions in tight-junction formation, likely resulting from an altered molecular composition of DM.On the other hand, deletion of Ntn4led to hyper-innervation of the corneal surface during development. When further characterizedin the context of wound recovery, adult Ntn4-/-mice showed a slightlyearlier reepithelialization, upregulated cell proliferation and reinnervation of wound area compared to wild-type littermates. Altogether, these data indicate that netrin-4 is likely a negative regulator of proliferative events in the cornea.Hownetrin-4 and laminin heterotrimers containing the laminin β2or γ3 subunits affect nerve-cell interactions in the cornea merits further inquiry. Given the observations presented herein, these molecules are important for corneal maintenance and have applications potential in strategies aimed toward treatment of corneal injuries, such as development of fully characterized, synthetic pro-regenerative membranes, or incorporation into implantable biomimetic scaffolds which can also mitigate current donor tissue shortages.
    • A TUMOR SUPPRESSOR FUNCTION FOR PTPN1 IN MYELOPROLIFERATIVE NEOPLASMS

      Mohi, Golam; JOBE, FATOUMATA (2016)
      Myeloproliferative neoplasms (MPNs) are a class of clonally-derived hematologic malignancies characterized by uncontrolled proliferation of myeloid lineage cells. Theyare classified into Philadelphiachromosome-positive (Ph+)MPNs, consisting of chronic myelogenous leukemia (CML), and Philadelphiachromosome-negative (Ph-)MPNs, consisting of polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis(PMF).The JAK2V617F mutation is the most common abnormality in Ph-MPNs, occurring in ~95% of PV patients, 55% of ET patients and 65% of PMF patients. JAK2V617F mutation results in constitutive activation of the JAK2 tyrosine kinase. Deletion of chromosome 20q (20q-) is a common chromosomal abnormality in myeloid neoplasms, including about 24%of MFcases. The 20q-lesion can coexist with JAK2V617Fmutation in MPN and MDS/MPN. The PTPN1gene is located on human chromosome 20q, within the commonly deleted region. PTPN1 is a tyrosine phosphatase and a known negative regulator of JAK-STAT signaling. The role of PTPN1 loss in the pathogenesis of MPNs and the mechanism by which loss of PTPN1 might contribute to various MPN phenotypes remains elusive. The goalsof this dissertation were to determine the effects of PTPN1 deficiency alone and in JAK2V617F-induced MPNs in vivo. To determine the mechanism by which PTPN1 mediates its tumor suppressor function using hematopoietic cells andassessment of PTPN1 status in 20q-MPN patients.Using conditional knock-out PTPN1 mouse model, we show that deletion of PTPN1 causes an MPN-like phenotype, characterized by increased WBC and NE counts and splenomegaly, compared to control mice. We also show that loss of PTPN1 causesfibrosis in older mice. PTPN1 knockdown significantly increased cell proliferation and activation of JAK2, MAPK and AKT signaling, whereas over expression in JAK2V617F-expressing cells attenuated cytokine-independent cell proliferation and signaling. Our data revealed a cooperative effect between PTPN1 deficiency and JAK2V617F expression in mice as shown by enhanced severity of theMPN phenotype and transformation to MF. Cell autonomous BMT revealed that the effects of PTPN1 deficiency are cell autonomous.Taken together, our results suggest a novel tumor suppressor function for PTPN1 in MPNs.
    • Design and Characterization of Proteins Rationally Engineered to Domain Swap by Mutually Exclusive Folding

      Loh, Stewart; Karchin, Joshua Michael (2016)
      Domain swapping is a mechanism for proteins to form dimers and higher order oligomers through the exchange of a section of their 3D structures. The backbone peptides of domain swapped oligomers are intertwined, but their 3D structures remain identical to their monomeric state, except for where they cross-over, termed a hinged region. We have developed a technique to engineer domain swapping interfaces with mutually exclusive folding (MEF). MEF achieves this by inserting a ‘lever’ protein into the surface loops of a host ‘target’ protein to form a target-lever fusion. This target-leveris conformationally strained with the lever and the target in a thermodynamic tug-of-war. When the lever is folded, the long distance between its N- and C-termini stretches apart the target and splits it in half. Conversely, when the target is folded, the short length of the loop where the lever was inserted compresses the lever and unfolds it. Domain swapping provides an escape from this tug-of-war as it allows the split target to refold and bypass the conformational strain. Because the lever is external to the target, adjusting the stability of the lever, through well-established thermodynamic principles, allows the propensity for domain swapping to be modulated without affecting the binding interface. This enables the design of “triggerable” levers which can reversibly induce domain swapping in response to a signal. Further, we can use domain swapping to turn the function of a target domain on and off. Two target-lever constructs are created with functional mutants in the target domain, one N-terminal to the lever and the other one C-terminal to the lever. Individually, both of these mutants are inactive, however if they are mixed and allowed to domain swap, then up to half of the target domains can swap out the functional mutations into the native active form. This bimolecular system in combination with induced domain swapping enables the design of modular bioswitches and biosensors.
    • MULTI-FUNCTIONAL EFFECTOR RESPONSES ELICITED FROM IgM MEMORY STEM CELLS

      Winslow, Gary; Kenderes, Kevin (2017)
      The response of memory B cells to challenge infection is fundamental to longterm protection against pathogens. Following challenge, memory B cells can rapidly differentiate into antibody-secreting cells (ASCs) to produce a secondary antibody response. Memory B cells have also been shown to re-enter into germinal centers and undergo additional rounds of affinity maturation. Both the isotype of the B cell and the signals that generated the B cell have been proposed to modulate how memory B cells respond. Initial studies proposed BCR-intrinsic factors are responsible for the differentiation of memory cells. IgM memory cells undergo differentiation in GCs following antigen challenge, while IgG memory cells rapidly differentiate into ASCs. Other studies found no link-between BCR isotype and differentiation. We investigated the differentiation of T-bet+ CD11c+ IgM memory B cells following challenge infection. IgM memory cells differentiated into IgM-producing plasmablasts. Other IgM memory B cells entered germinal centers, underwent class switching, and became switched memory cells. Yet other donor cells were maintained as IgM memory cells. The IgM memory cells also retained their multi-lineage potential following serial transfer. The kinetics of the IgM memory response mimicked the kinetics of the primary response. Thus, IgM memory cells can differentiate into all effector B cell lineages, and undergo self-renewal, properties that are characteristic of stem cells; however, differentiation occurs with the same kinetics of the primary response. We propose that memory B cells have varying degrees of stem cell likeness. IgM memory stem cells retain the most differentiating capacity but respond to challenge similarly to naïve cells, while IgG effector memory cells are primed to rapidly differentiate into IgG ASCs.