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Recent Submissions

  • Localization of sarcomeric proteins during muscle assembly in mouse cardiomyocytes and skeletal myotubes

    Sanger, Jean; Sanger, Joseph; Welchons, Matthew J (2017)
    This study seeks to investigate the role of contractions in myofibrillogenesis, and structure of nascent myofibrils. The model system employed in these experiments was cultured quail myotubes. In order to determine the role of contractions in myofibrillogensis, contractions were indirectly inhibited with elevated KCl, and directly inhibited with 2,3 butanedione monoxime (BDM), a small cell-permeable inhibitor of actin & muscle myosin interactions. Myotubes were treated with contraction inhibitors at 2½days –soon after the main fusion event. On the 6thculture day, there was significant delay in myofibrillogensis in myotubes exposed to elevated KCl. This delay was characterized by the expansion of nascent myofibrils at the spreading edges of myotubes. This delay in myofibrillogenesis was not accompanied by diminished accumulations of muscle myosin. On the 4thculture day, there was complete arrest of myofibrillogenesis at the nascent step with the treatment of BDM. As a result, it is concluded that contractions are necessary for the progression of nascent myofibrils to mature myofibrils. The structure of nascent myofibrils was further investigated with super resolution microscopy. Structured illumination microscopy (SIM) and stimulated emission depletion (STED) microscopy revealed mini-A-bands –shorter than 1.5 μmin length –associated with nascent myofibrils. These structures were spaced at varying intervals, and oriented at angles deviating the from the actin superstructure of the nascent myofibril. Mini-A-bands were also observed to progressively in expand in length distal to the spreading edges of myotubes. STED imaging indicates that some mini-A-band are adjacent to, and not integrated within, the actin superstructure of nascent myofibrils.
  • Metabolic Control of Autoimmunity Through Autophagy

    Choudhary, Gourav (2019)
    Metabolism plays a key role in immune cell activation and differentiation. Immune cell activation depending on their biosynthetic and bioenergetic needs leads to profound metabolic reprograming. Proinflammatory subsets of immune system cells such as effector T cells show dependency on glycolysis, whereas, regulatory T cells rely on oxidative phosphorylation. Under metabolic stress, immune cells utilize autophagy to overcome nutrient scarcity, an alternate method of recycling amino acids and other metabolic precursors. Limitation of nutrients such as amino acids activates mechanistic target of rapamycin (mTOR) in the immune cells. mTOR acts as a metabolic mediator, associated with mitochondria and metabolic needs of the immune cells. Homeostasis between mTOR activation and autophagy decides the fate and functionality of specific immune cells. The activation of mTOR is widely acknowledged in the pathogenesis of SLE, whereas, autophagy has been linked with antigen processing, presentation, and immunoregulation. In this study, we focused on Rab4A, an endosomal GTPase and Transaldolase, a rate limiting enzyme of the pentose phosphate pathway (PPP). Rab4A is over expressed in SLE T cells and facilitates lysosomal degradation of CD4 and CD3. Transaldolase is also overexpressed in T cells from SLE patients and SLE prone mice. First, we examined the role of Rab4A in a pristane-induced mouse model of SLE. Since Rab4A protects from pristane-induced alveolar lung hemorrhage, we tested the hypothesis that Rab4A will also protect from pristane-induced lupus nephritis. We found that overexpression of a constitutively active form of Rab4A limits antinuclear antibody production. Further, we found that Rab4A protects from pristane-induced renal injury by restricting immune complex depositions in the kidney. In additions, we found that Rab4A abrogates kidney-infiltration by lymphocytes and protects from podocyte injury. Furthermore, Rab4A facilitates the lysosomal mediated activation of mTOR. Possibly, the Rab4A mediated activation of mTOR in regulatory T cells leads to suppression of pristane-induced pro-inflammation signaling. In the second part, we investigated if aldose reductase (AR) deficiency can protect from Transaldolase mediated pathogenesis of liver disease. We found a coordinated regulation between AR and TAL, leading to the disease progression.
  • DISCOVERY OF A ROLE OF FMRP IN R-LOOP REGULATION AND GENOME MAINTENANCE THROUGH BREAK-SEQ ANALYSIS OF THE FRAGILE X GENOME

    Chakraborty, Arijita (2020)
    Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMRP translation regulator 1 (FMR1) gene and deficiency of its product, FMRP. FMRP is known as a translation repressor whose nuclear function is poorly understood. We investigated the global impact on genome stability due to FMRP loss. We applied Break-seq to a human cell line-based model for FXS and mapped genome-wide spontaneous and replication stress-induced DNA double strand breaks (DSBs) for the first time. We report that the genomes of FXS patient-derived cells are inherently unstable and accumulate more than twice as many DSBs as those fromnormal cells. The DSBs in FXS cells are enriched in neuron projection and synapse organization pathways. We further demonstrate that replication stress-induced DSBs in FXS cells correlate with R-loop forming sequences. FMRP, and not an RNA-binding mutant FMRP-I304N, abates R-loop-induced DSBs during programmed replication-transcription conflict.Moreover, exogenously expressed FMRP in FXS patient-derived cells reduces the replication stress-induced DSB formation. We conclude that the FXS cells are more susceptible to DNA replication stress. Furthermore, we identified chromatin binding sites of FMRP for the first time in human lymphoblastoid cells.Through mapping FMRP-bound chromatin loci in normal cells and correlating with FX-specific chromosome breaks, we identified novel FXS-susceptible genes. We show that FX cells have reduced expression of the uridine diphosphoglucuronosyl transferase 1 family enzymes, suggesting defective xenobioticmetabolism. In addition, using transcriptome analysis, we show that DNA repair genes are downregulated in FX cells under replication stress. Finally, we report a direct binding interaction between FMRP and R-loop and that the C-terminal domain is important for this interaction. Therefore, we proposethat FMRP is a novel genome maintenance protein required for preventing R-loop formation during replication stress. Our study provides newinsights into the etiological basis for FXS.
  • A STRING OF LIGHTS: MATURIN EXPRESSION AND POTENTIAL ROLE DURING MOUSE RETINOGENESIS

    Ly, Christine (2020)
    During retinal development, a pool of progenitor cells divides to generate daughter cells that eventually differentiate into the seven retinal cell types, including horizontal cells (HCs) and retinal ganglion cells (RGCs). Much about how cells exit the cell cycle and maintain a differentiated state remain unknown. Dysregulation of this process can alter the cellular composition and function of the retina.Thus, by studying this developmental process, we can better understand the mechanisms by which progenitor cells become functional, differentiated cells. Our previous work determined that Maturin (Mturn)is highly conserved in its expression pattern and protein sequence across various vertebrate species. Furthermore, we concluded that it is required for differentiation of primary neurons in Xenopuslaevis. Preliminary work in mice revealed that in the absence of Mturn, extensive folds occur in the retina. I used this model to characterize the expression of Mturnin the mouse retina and ask if Mturn is required for normal mice retinogenesis. By immunostaining retinal sections with various cell type-specific antibodies, I found that Mturn is expressed in differentiating cells and not in proliferating cells. In addition to determining that its expression is maintained in mature HCs, I concluded that Mturn is not required for generating the proper number HCs. Our results from studies on Mturn in both frogs and mice have led us to hypothesize that Mturn may function to maintain HCs in differentiated state and prevent their reentry into the cell cycle. Although preliminary experiments testing this hypothesis were inconclusive, future work should continue to investigate the role of Mturn in retinogenesis.
  • JUNCTIONAL ARMADILLO (β-CATENIN) MAINTAINS PROPER TISSUE ARCHITECTURE DURINGDROSOPHILAEYE DEVELOPMENT

    Pignoni, Francesca; DeSantis, Dana F (2020)
    Formation of thecompoundeye of Drosophilarequires carefully orchestrated developmental events that occur in its progenitor epithelium, the eye imaginal disc.This tissue is composed of two continuous, apposed epithelia: the disc proper epithelium (DpE), which forms the retina, and the peripodial epithelium (PE), which ultimately forms head cuticle. In this work, I describe an armadillo (b-catenin)loss-of-function condition in which the developing DpEis disrupted and displays a phenotype that I call“retinalshift”. This developmental phenotype ultimately results in abnormal fly eye morphology that is incompatible with compound eye vision.I uncover a role for the PE in maintaining proper retinal epithelium morphology during eye formation and trace the molecular mechanism to the regulation of Hippo-Yki pathway in PE cells by the function of Armadillo at the adherens junctions.
  • DIRECTING DENDRITOGENESIS: DEFINING THE ROLE OF REELIN AND CSPGS IN THE CONTROL OF CORTICAL DENDRITE FORMATION

    Olson, Eric; Zluhan, Eric (2020)
    Appropriate dendritic development is essential for normal neuronal function throughout life. Disruptions in neuronal dendrite structure alter brain circuitry and are associated with debilitating neurological disorders.The Reelin signaling pathway is critical for proper cortical dendrite orientation and outgrowth. In Reelin null cortices (reeler), dendrites are unstable, retracting from and avoiding their normal target region called the marginal zone (MZ). This observation raises the possible existenceof a dendritic destabilizing cue in the MZ that can be counteracted by Reelin-signaling.The MZis cell sparse but highly enriched in chondroitin sulfate proteoglycans (CSPGs). While CSPGs are known to inhibit axonal outgrowth, their impact on dendritic growth is unclear.Here, we demonstrate that the growth of the apical dendrite is also inhibited by CSPGs. Soluble CSPGs and CSPG-patterned stripes are inhibitory to dendrite growth, butthis inhibitory effect can be reversed by CSPG ablation via chondroitinase treatment and by activation of the Reelin signaling pathway. In reeler explants, chondroitinase treatment rescues dendrite growth into MZ. Prior studies have shown that the serine threonine kinase Akt is essential for Reelin-dependent dendritic growthand also functions in CSPG-dependent neurite retraction. We find that CSPGs induce Akt dephosphorylation which isreversed by Reelin addition. CSPG presence had no effect on the cytoplasmic adaptor Dab1, which is rapidly phosphorylated in response to Reelin. Dab1-deficient neurons were sensitive to CSPG stimulation, but Reelin-dependent phosphorylation was blunted. This suggests that the extracellular signals imparted on dendrites by Reelin and CSPGs at the MZ converge intracellularly downstream of Dab1 atthe level of Akt to regulate dendritogenesis in the MZ.Disruptions in Reelin signaling cause intellectual disability and have been linked to autism. Thus, these findings identify a context in which Reelin signaling operates and provide insight into the underlying mechanism of neurodevelopmental disorders.
  • Ethanol-Induced Effects of the Microtranscriptome on Natural Gene Expression

    Middleton, Frank; Ignacio, Cherry Mae Gonzalez
    Reliable, minimally invasive biomarkers that predict the extent of alcoholism-induced CNS damage are currently lacking. This limits the selection of rational interventions and hampers the ability to gauge therapeutic effects. Developing biomarkers that indicate early CNS damage may prove useful in deterring the emergence of alcohol use disorders (AUDs). Extracellular microRNAs (miRNAs) can be informative molecular indicators of neuronal gene expression alterations. They repress large fractions of protein-coding genes and are highly-involved in intercellular signaling between both proximal and distal neurons. This work has focused on (1) examining whether extracellular miRNAs in the serum of individuals diagnosed with AUDs can be used as biomarkers of alcohol-induced brain damage, (2) determining in vivo the ethanol-inducedeffects imparted by miRNAs and their targets in the brain, (3) evaluating their role ininterventions that can reverse behavioral impairment and (4) testing the ability of extracellular miRNAs to transfer ethanol-induced pathologies to ethanol-naive cells. There are five major findings from this work. First, two independent quantification technologies demonstrated comparable differences in miRNA expression levels betweenAUDs and controls and revealed significant correlations between candidate miRNA biomarkers and medical, neuroimaging and drinking parameters. Second, in rats manymiRNAs significantly altered by ethanol in the hippocampus following maternal or postnatal exposure were also changed in the serum. Moreover, postnatal consumption activated cell-cycle pathways in the hippocampus while maternal exposure affected unfolded protein response pathways in adolescent offspring. Third, the lack of social motivation seen following fetal exposure was reversed as a result of social enrichment. Analysis of the integrated data in the amygdala and ventral striatum revealed several functional gene networks whose activation patterns following fetal ethanol exposure were reversed by social enrichment. Fourth, transfer of purified exosomes from ethanol-exposed to ethanol-naive cells conferred many gene expression changes consistent with ethanol exposure. Lastly, examination of all the data revealed consistent changes in miRNAs that independently converged on cell death, cell proliferation and cell cycle regulatory processes, regardless of the species, paradigm and source. The findings in this work illustrate the utility of miRNAs as peripheral biomarkers of AUDs and suggest novel epigenetic mechanisms affected by alcohol.
  • Rab4acontrol over metabolism and mTOR drives disease progression in Systemic Lupus Erythematosus

    Perl, Andras; Huang, Nick (2020-05-15)
    Endosomal trafficking is key to intercellular communication and metabolic regulation of immunological development. Rab4a, an endosomal trafficker, is elevated in lupus T cells and polymorphisms of the Rab4a gene have been linked to disease susceptibility. Here, we report the constitutive activation of Rab4a increases susceptibility and severity to lupus nephritis in the genetic SLE1.2.3. model of lupus and is corrected by the deletion of Rab4a in T cells. Alternatively, in a pristane model of induced autoimmunity, the deletion of Rab4a in T cells magnifies the pulmonary manifestations of diffuse alveolar hemorrhage that is otherwise protected by the constitutive activation of Rab4a. Rab4a mediates these changes through control over mTOR, mitochondrial function and homeostasis, and immunological development. In particular, inactivation of Rab4a in T cells reduces expression of activation signals, mitochondrial mass and electrochemical potential. Alterations to Rab4a activity drives the aberrant development and function of anti-inflammatory regulatory T cells and pro-inflammatory double-negative T cells. These data provide new insights into the regulation of metabolism and immunological development through endosomal trafficking. As such, the targeting of Rab4a is a novel therapeutic approach in the treatment of autoimmune diseases such as lupus, which has lacked new targeted therapeutics for more than half a century.
  • REGULATION AND FUNCTION OF TUMOR SUPPRESSOR ECRG2 IN RELATION TO DNA DAMAGE AND MICROTUBULE DYNAMICS IN HUMAN MALIGNANCIES

    Huang, Ying; Patel, Harsh (2020)
    phageal Cancer-Related Gene 2 (ECRG2) is a novel tumor suppressor which is frequently mutated or downregulated in multiple human cancers. Previous studies have demonstrated that ECRG2 inhibits growth of cancer cells by inducing apoptotic death. However, the molecular basis of its regulation and involvement in DNA damage response remain to be elucidated. The function of tumor suppressor p53 in cellular response to stress conditions, such as DNA damage, has been well-established. In the present study, we report for the first time, that ECRG2 is a novel pro-apoptotic transcriptional target of p53 and ECRG2 expression is induced by DNA damage in a p53-dependent manner. Moreover, we demonstrate that disruption of ECRG2 leads to reduced apoptosis and improved survival following the treatment with DNA damage-inducing anticancer agent despite p53 activation in cancer cells. Significantly, we characterized a natural variant in ECRG2promoter (rs3214447) that is found in the genomes of ~38.5% of world population and showed that ECRG2 promoter with rs3214447 variant is defective in responding to p53 and DNA damage. Thus, ECRG2 is an important executor of p53-mediated apoptosis in response to DNA damage. We also report a novel biological function of ECRG2 and demonstrate that ECRG2 interacts with and stabilizes microtubules. ECRG2 was shown to protect the microtubules against the destabilization induced by cold and nocodazole treatment. In addition, we show that ECRG2 increases acetylation of microtubules, which is associated with more stable microtubules. Importantly, we demonstrate that ECRG2 disruption give rise to increased cell proliferation by elevated activation of Akt. Taken together, our findings ascribe a novel function to ECRG2 in the regulation of microtubule dynamics and cancer cell proliferation. ECRG2-mediated tumor suppressor activities elucidated in this dissertation are clinically significant. Our database analyses reveal that cancer patients with lower ECRG2expression in their tumors had poor prognosis and reduced disease-free survival as compared to their counterparts. These observations suggest that loss of ECRG2 expression and function confers survival advantage to cancer cells. Collectively, this dissertation highlights novel aspects of ECRG2 regulation and function in cancer cell sensitivity to DNA damage-inducing anticancer therapy, microtubule dynamics and cell proliferation.
  • NEAR-ATOMIC RESOLUTION STRUCTURE OF THE YEAST VACUOLAR (V-) ATPASE MEMBRANE SECTOR Vo IN LIPID NANODISC

    Wilkens, Stephan; Stam, Nicholas J (2020-04-10)
    Vacuolar ATPase (V-ATPase) is a large multisubunit enzyme that acidifies subcellular organelles and the extracellular space. Its activity is regulated by reversible disassembly, causing V-ATPase dissociation into soluble V1-ATPase and membrane-integral Voproton channel sectors.The goal of this thesis project was to observethe yeast Voin a physiologically relevant, auto-inhibited state, i.e. in its form dissociated from the ATPase sector, V1, in order to better visualizethe closed pore and to identify testable hypotheses on why the pore remains closed following dissociation from V1. Towards this aim we present two chapters:In Chapter 1, we detail a single-particle negative stain EM study of lipid nanodisc reconstituted Vo,which suggesteddissociated Vois halted in theso-called rotational state 3 of the holo-enzyme.We performed site directed mutagenesis and binding studies of subunits aand dto test and validate this hypothesis.In Chapter 2,we further detaillipid nanodisc reconstitutedVoin a high-resolutioncryoEM structure,confirmingour earlier identification of Voresting in rotational state 3, andproviding structural information of the sectorat the amino acid level. Through this work we proposed apossible mechanism for transmembrane proton transport in the V-ATPaseandidentifieda new subunit member of Vo, assembly factor Voa1.The studies shown here highlight the potential of lipid nanodisc reconstitution of membrane protein complexes, give insight into a conformational mismatch between autoinhibited V1and halted Vowith the implication that the mismatch may serve to prevent unintended reassembly of V-ATPase upon activity silencing, and propose a chemical basis for transmembrane proton transport in the Voproton pore.
  • Molecular Analysisof Saccharomyc escerevisiae RNA Polymerase I Core Factor Complex and its Interaction with Promoter DNA

    Knutson, Bruce; Jackobel, Ashleigh J (2020)
    Gene transcription and protein synthesis are essential molecular processes required for all living organisms. In eukaryotes, messages encoded within DNA are transcribed by three DNA-dependent RNA polymerases (Pols I-III) into ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA), respectively. General transcription factors (GTFs) help recruit Pols to their appropriate gene promoters as well as facilitate template opening and transcription start site (TSS) selection. In Saccharomyces cerevisiae, the Pol I pre-initiation complex (PIC) is formed by numerous GTFs that include Upstream Activating Factor (UAF), Core Factor (CF), TATA-binding protein (TBP), and Rrn3. This unique set of GTFs engage ribosomal DNA (rDNA) through interactions with regulatory elements of the promoter known as the Upstream Activating Sequence (UAS) and the Core Element (CE). Here, we resolve the cryo-electron microscopy (cryo-EM) structure of CF bound to the rDNA promoter at 3.8Å near-atomic resolution and determine itsDNA binding preferences in which CF preferentially binds to the GC-minor groove. Briefly, our cryo-EM studies reveal that the CF-DNA interaction is mediated by two CF subunits, Rrn7 and Rrn11. We also found that the path of promoter DNA is relatively straight in the Pol I PIC, which is strikingly different from the bent promoters observed in structures of the Pols II/III PICs. We identified three states of CF engagement with promoter DNA (States 1-3) in which CF acts as a ratchet toforceDNA into the active site of the polymerase that facilitates the melting of the double-stranded DNA template in an ATP-independent manner, another unique feature of the Pol I system. Using in vitroDNA binding assays, we have identified a 12 base pair (bp) region within the CE that is necessary and sufficient for CF binding. We have also demonstrated that the human anticancer compound CX-5461 can inhibit yeast cell growth and blocks yeast CF binding to both yeast and human rDNA promoters in vitro. Furthermore, we show that the human Core Promoter Element (CPE) can functionally replace the yeast CE in a position-dependent manner. Together, these results reveal the unique molecular architecture of the Pol I PIC and suggest a conserved sequence-independent binding mechanism of CF with promoter DNA.
  • Effects of Rab4A Mutations on Mouse Behavior, mTORC1 Activity,and Surface 8Receptor/TransporterRecycling

    Perl, Andras; Winans, Thomas C (2020-10-03)
    Through studying endosomal regulation, I found that a single amino acid 47mutation (Q72L) in the Rab4A gene leads to neurological disorders in two separate 48mouse strains. In the C57Bl/6 (SLE(WT)) background,I found thatknock-in the 49Rab4A gene leads to hyperactivity, which resembles both autism spectrum 50disorders (ASD) and attention-deficit hyperactivity disorder (ADHD). On a lupus-51prone background (SLE(1.2.3)) I found thatthe same mutation led to hypoactivity, 52which indicates a more severe neuropsychiatric systemic lupus erythematosus 53(NPSLE) than SLE(1.2.3)mice with wild type Rab4A.54The same mice were studied in chapter two, where mTORC1 activity was 55confirmed to be elevated in CD4+ T cells when Rab4A was knocked-in (Rab4A(KI))56compared to Rab4A(WT) cells.In young mouse brains prior to disease onset, I found57increases of mTORC1 and oxidative stress in Rab4A(KI) brains relative to 58Rab4A(WT) brains. In the same brains, there was also a depletion of GLUT1 and 59IFNGR1.Many of these changes were absent in the adult mice, after disease had 60developed.61SLE(1.2.3) mice with the three Rab4A alleleswere treated withrapamycin or 62NAC,and brains were collected.In these brains, there wasevidence that the 63hypoactive Rab4A(KI) SLE(1.2.3) had lower mTORC1 activity than Rab4A(WT) and 64Rab4A(KO) mice.This finding indicatesdepression, which is a pattern seen in major 65depressive disorder(MDD). Depression is also a symptoms of NPSLE. Interestingly, 66rapamycin increased mTORC1 activity in theRab4A(KI)brains compared Rab4A(KI) 67mice treated with vehicle, indicating a positive effect from the drug.
  • CD11C+ T-BET+ B CELLS IN INFECTION AND AUTOIMMUNITY

    Winslow, Gary; Levack, Russell (2020)
    CD11c+ T-bet+ B cells serve crucial roles in both protective immunity and autoimmunity.However, the ontogeny of these cells remains unclear, and strategies to target them in vivo have yet to be identified. Here, we demonstrate that developing CD11c+ T-bet+ B cells received help in the form of IL-21, IFN-γ, and CD40L from a population ofT follicular helper 1(TFH1)cells outside of formal germinal centers (GC). These TFH1cells provided help to developing CD11c+ T-bet+ B cells in two distinct phases: IFN-gwas provided early following infection, and CD40L was provided later. Unlike the TFH1cells, CD11c+ T-bet+ B cells required the GC-associated transcription factor Bcl-6 for their development, but not T-bet. While the CD11c+ B cells that arose in the absence of T-bet appeared nearly identical to their T-bet-competent counterparts,they did not switch to IgG2c. These data support a model where, in the absence of formal GCs, TFH1cells provide GC-like help to developing CD11c+ T-bet+ B cells and while T-bet is not required for the development of these T-bet+ B cells,it is required for appropriate class-switch recombination (CSR). Our work also demonstrates that mature CD11c+ T-bet+ B cells, which arise in both immunity and autoimmunity,wereeliminated following treatment with the adenosine 2a receptor (A2aR) agonist CGS-21680. Depletion of these CD11c+ T-bet+ B cells occurred in a B cell-intrinsic manner and was corelated with improved disease outcome in a mouse model of lupus. Preliminary data indicated that human CD11c+ B cells expressed the A2aR,and these cells were depleted following CGS-21680 treatment in vitro, suggesting that A2aR-agonistadministrationmay also be effective in the treatment of human autoimmune diseaseswhere CD11c+ Bcell play a role. Overall, this work provides novel insight into the development of T-bet+ B cells and identifies the first pharmacological approach to target these cells in vivo.
  • Liver-specific glucocorticoid action in alcoholic liver disease

    Lu, Hong; Wang, Yazheng (2021)
    The number of deaths due to alcoholic liver disease is increasing every year. Glucocorticoids (GCs) are the only first-line drugs for alcoholic hepatitis (AH) treatment but have limited efficacy. Long-term high-dose GC use can cause various side effects on extrahepatic tissues, such as immunosuppression and neuromuscular side effects, which may be a limiting factor for GC treatment of AH. Therefore, liver-specific GC-targeted therapy may have multiple advantages compared with systemic GC for AH. This research explored the role of liver-specific deficiency of glucocorticoid receptor (GR) in AH induced by a high-fat diet (HFD) plus ethanol binge. Females are less prone to AH induced by HFD plus acute binge drinking, likely due to sex differences in estrogen (E2) signaling. We found that hepatic GR deficiency worsened steatosis in both genders of AH mice but only aggravated the liver injury in male AH mice. Multiple signaling pathways were dysregulated in GR knockout AH mice. Interestingly, hepatic expression of estrogen receptor (ERα) was induced, and the E2-inactivating enzyme was markedly down-regulated in GR knockout AH mice, suggesting enhanced E2 signaling in these mice. Our data mining found marked dysregulation of many GR-target genes important for lipid catabolism, cytoprotection, and inflammation in patients with severe AH. These key GR-target genes were similarly induced or down-regulated by our liver-targeting GC prodrugs and the parent drug at 1μM in primary human hepatocytes. In contrast, GC prodrugs had much weaker inhibitory effects than the parent drug on LPS-induction of IL-1B in mouse macrophages, suggesting a good liver selectivity of our liver-targeting GC prodrugs. The ultimate goal of this study is to determine the mechanistic role of GR in alcoholic fatty liver disease and develop targeted drug therapies to treat alcoholic hepatitis.
  • THE TUMOR SUPPRESSOR FOLLICULIN REGULATES GLYCOLYSIS BY SPECIFICALLY BINDING AND INHIBITING LACTATE DEHYDROGENASE-A

    Mollapour, Mehdi; Woodford, Mark R. (2021)
    Folliculin (FLCN) is tumor suppressor protein whose function remains a topic of debate. Germline mutations in FLCN predispose affected individuals to develop Birt-Hogg-Dubé syndrome, which is characterized by facial fibrofolliculomas, pulmonary cysts, spontaneous pneumothorax, and renal cell carcinoma. These kidney tumors exhibit an elevated glycolytic phenotype even in the presence of oxygen, an observation commonly known as the “Warburg effect.” This phenomenon is driven by the activity of lactate dehydrogenase-A (LDHA), the enzyme responsible for the interconversion of pyruvate and lactate in the terminal step of glycolysis. Our work herein shows that FLCN is a specific intracellular inhibitor of LDHA. Biochemical and biophysical analyses mapped the interaction and inhibition of LDHA activity to an unstructured loop in FLCN positioned between the amino and carboxy-terminal domains. Characterization of a minimal 10-amino acid FLCN-derived peptide demonstrated that it was sufficient to both bind and inhibit LDHA activity in vitro. Further, treatment of FLCN-deficient cells with this FLCN-derived peptide is sufficient to suppress glycolysis. Interestingly, evaluated cancer cell lines derived from solid tumors of the lung, breast, prostate, bladder, and colon also demonstrate dysregulation of LDHA activity and dissociation of FLCN-LDHA interaction. Previous work has shown that inhibition of LDHA in cancer cells leads to apoptosis. Accordingly, treatment of these cell lines with the FLCN-peptide results in apoptosis, suggesting a potential therapeutic intervention. Taken together, inhibition of LDHA by the tumor suppressor FLCN provides a mechanistic explanation for the endogenous regulation of glycolysis.

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