• The p53-Zn2+ Energy Landscape and Metallochaperone Hypothesis

      Loh, Stewart; Blanden, Adam (2017)
      p53 is a tumor suppressor protein found mutated in essentially half of human cancers, and dysfunctional in nearly all human cancers. Each DNA-binding domain of the protein contains a critical tetrahedrally coordinated Zn2+. In this work, we present a quantitative thermodynamic model describing the energetics of the p53-Zn2+ interaction, as well as the mechanism of action of a new class of therapeutic compounds we call synthetic zinc metallochaperones (ZMC) that restore proper structure and function to many mutant p53s by delivering Zn2+ to the protein in the cell. We combine recombinant protein expression and in vitrobiophysical characterization with cell biology, molecular biology, medicinal chemistry, and live cell imaging to address these issues. Our model for both the mechanism of action of ZMCs and the p53-Zn2+interaction are broadly based on the Metallochaperone Hypothesis originally proposed by our group in 2010. We find that the core tenants of the Metallochaperone Hypothesis are accurate, and have expanded that model to quantitatively describe the link between p53-Zn2+ binding and protein stability noted for decades in the field. We find that at physiological temperature and Zn2+ concentrations, wild-type p53 has a folding energy of ~0 kcal mol-1, and as such is exquisitely sensitive to inactivation by mutation, and rapidly changes the fraction folded in response to changes in Zn2+ concentration. We demonstrate that ZMCs are ionophores, transport Zn2+ from the extracellular space into cells, and rescue mutant p53 by increasing the intracellular free Zn2+ concentration. This increase in Zn2+ stabilizes the mutant proteins via the same mechanism previously described for substrate stabilization of enzymes, and is only seen in a "Goldilocks Zone" of Zn2+ concentrations and ZMC Kds. This presents a fundamentally new way to interact with and reactivate mutant p53s, and raises questions about the potential for biological exploitation of this interaction for signaling or other functions.
    • Patterns of Psychopathology and Dysfunction in High-Risk Children of Parents With Panic Disorder and Major Depression

      Biederman, Joseph; Faraone, Stephen V.; Hirshfeld-Becker, Dina R.; Friedman, Deborah; Robin, Joanna A.; Rosenbaum, Jerrold F. (American Psychiatric Association Publishing, 2001-01)
      Objective: The purpose of the study was to evaluate 1) whether an underlying familial predisposition is shared by all anxiety disorders or whether specific risks are associated with specific disorders, and 2) whether panic disorder and major depression have a familial link. Method: The study compared four groups of children: 1) offspring of parents with panic disorder and comorbid major depression (N=179), 2) offspring of parents with panic disorder without comorbid major depression (N=29), 3) offspring of parents with major depression without comorbid panic disorder (N=59), and 4) offspring of parents with neither panic disorder nor major depression (N=113). Results: Parental panic disorder, regardless of comorbidity with major depression, was associated with an increased risk for panic disorder and agoraphobia in offspring. Parental major depression, regardless of comorbidity with panic disorder, was associated with increased risks for social phobia, major depression, disruptive behavior disorders, and poorer social functioning in offspring. Both parental panic disorder and parental major depression, individually or comorbidly, were associated with increased risk for separation anxiety disorder and multiple (two or more) anxiety disorders in offspring. Conclusions: These findings confirm and extend previous results documenting significant associations between the presence of panic disorder and major depression in parents and patterns of psychopathology and dysfunction in their offspring.
    • Pediatric mania: a developmental subtype of bipolar disorder?

      Biederman, Joseph; Mick, Eric; Faraone, Stephen V.; Spencer, Thomas; Wilens, Timothy E; Wozniak, Janet (Elsevier BV, 2000-09)
      Despite ongoing controversy, the view that pediatric mania is rare or nonexistent has been increasingly challenged not only by case reports, but also by systematic research. This research strongly suggests that pediatric mania may not be rare but that it may be difficult to diagnose. Since children with mania are likely to become adults with bipolar disorder, the recognition and characterization of childhood-onset mania may help identify a meaningful developmental subtype of bipolar disorder worthy of further investigation. The major difficulties that complicate the diagnosis of pediatric mania include: 1) its pattern of comorbidity may be unique by adult standards, especially its overlap with attention-deficit/hyperactivity disorder, aggression, and conduct disorder; 2) its overlap with substance use disorders; 3) its association with trauma and adversity; and 4) its response to treatment is atypical by adult standards. Biol Psychiatry 2000;48: 458–466 © 2000 Society of Biological Psychiatry.
    • Pediatric mania: a developmental subtype of bipolar disorder?

      Biederman, Joseph; Mick, Eric; Faraone, Stephen V.; Spencer, Thomas; Wilens, Timothy E; Wozniak, Janet (Elsevier BV, 2000-09)
    • PHOSPHORYLATION AND UBIQUITINATION REGULATE PROTEIN PHOSPHATASE 5 ACTIVITY AND ITS PROSURVIVAL ROLE IN KIDNEY CANCER

      Mollapour, Mehdi; Dushukyan, Natela (2018)
      Protein Phosphatase 5 (PP5) is a serine/threonine phosphatase known to regulate many essential cellular functions including steroid hormone signaling, stress response, proliferation, apoptosis, and DNA repair. PP5 is a knownco-chaperone of the molecular chaperone heat shock protein 90 (Hsp90), and its regulation of Hsp90aidswiththe proper activation of Hsp90 clients and withsteroid hormone signaling.Hsp90 is also one of the strongest activators of PP5, as it releases the auto-inhibition of PP5 by interacting with the N-terminal tetratricopeptide repeat (TPR) domain of PP5. Our lab has recently shown that PP5 is phosphorylated at T362, and that this phosphorylation acts as an “on switch” resultingin the hyperactivation of PP5. Misregulation of this key phosphatase has been shown to aid in the tumor progression of ER-dependent and independent breast cancer. Elevated PP5 levels have also been linked to colorectalcancer, hepatocellular carcinoma (HCC), lymphoma, and prostate cancer. The work presented here reveals the pro-survival role that PP5 plays in kidney cancer. Clear cell renal cell carcinomas (ccRCC) are most often driven by mutations in the von Hippel-Lindau tumor suppressor (VHL). The data in this thesis shows that VHL binds and multi mono-ubiquitinates PP5 at two lysine residues K185 and K199. This post-translational modification negatively regulates PP5 likean “off switch” and ultimately leads to its degradation bythe proteasome. Mutations in the VHLgene that result in inactive mutants or a lack of VHL protein expression lead to ccRCC tumors. The data in this thesis shows that these VHL-nulltumors become dependent on elevated levels of PP5, and that both PP5 knockdown and inhibition lead to cancer cell death. The data further shows that the decrease in PP5 activity in VHL-null cells results in the induction of the extrinsic apoptotic pathway with a dramatic increase in the cleavage of PARP and caspases 3, 7, and 8.
    • Placebo and nocebo responses in randomised, controlled trials of medications for ADHD: a systematic review and meta-analysis

      Faraone, Stephen V.; Newcorn, Jeffrey H.; Cipriani, Andrea; Brandeis, Daniel; Kaiser, Anna; Hohmann, Sarah; Haege, Alexander; Cortese, Samuele (Springer Science and Business Media LLC, 2021-05-10)
      The nature and magnitude of placebo and nocebo responses to ADHD medications and the extent to which response to active medications and placebo are inter-correlated is unclear. To assess the magnitude of placebo and nocebo responses to ADHD and their association with active treatment response. We searched literature until June 26, 2019, for published/ unpublished double-blind, randomised placebo-controlled trials (RCTs) of ADHD medication. Authors were contacted for additional data. We assessed placebo effects on efficacy and nocebo effects on tolerability using random effects metaanalysis. We assessed the association of study design and patient features with placebo/nocebo response. We analysed 128 RCTs (10,578 children/adolescents and 9175 adults) and found significant and heterogenous placebo effects for all efficacy outcomes, with no publication bias. The placebo effect was greatest for clinician compared with other raters. We found nocebo effects on tolerability outcomes. Efficacy outcomes from most raters showed significant positive correlations between the baseline to endpoint placebo effects and the baseline to endpoint drug effects. Placebo and nocebo effects did not differ among drugs. Baseline severity and type of rating scale influenced the findings. Shared non-specific factors influence response to both placebo and active medication. Although ADHD medications are superior to placebo, and placebo treatment in clinical practice is not feasible, clinicians should attempt to incorporate factors associated with placebo effects into clinical care. Future studies should explore how such effects influence response to medication treatment. Upon publication, data will be available in Mendeley Data: PROSPERO (CRD42019130292).
    • 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.
    • POST-TRANSLATIONAL REGULATION OFCO-CHAPERONES AFFECTS HSP90 DRUG SENSITIVTY IN CANCER

      Mollapour, Mehdi; Dunn, Diana (2017)
      Heat Shock Protein-90 (Hsp90) is a molecular chaperone critical to thestability and activity of over 200 proteins known as “clients” including many oncogenes. Hsp90 chaperone function is linked to its ability to hydrolyze ATP and Hsp90 drugs inhibit its activity leading to the degradation of clients, thus making Hsp90 an attractive target for cancer therapy. The Hsp90 chaperone cycle is fine-tuned by another group of proteins called co-chaperones. They modifythe cycle, allowing Hsp90 to chaperone different pools of clients. Post-translational modifications (PTM) of Hsp90 and its co-chaperones can also regulate the chaperone cycle, and affect Hsp90 drug sensitivity. Here it is shownthat c-Abl kinase phosphorylates Y223in the co-chaperoneAha1, promotingits interaction with and stimulation of Hsp90 ATPase activity. Pharmacologic inhibition of c-Abl prevents the Aha1-Hsp90 interaction thereby, hypersensitizing cancer cells to Hsp90 inhibitors.Another co-chaperone of Hsp90, protein phosphatase-5 (PP5), mediates thede-phosphorylation of the co-chaperone Cdc37which is an essentialprocessfor the activation of kinase clients. The crystal structure of phospho-Cdc37 bound to the catalytic domain of PP5revealed elements of substrate specificity within the phosphatase cleft. Hyperactivityandhypoactivity of PP5 increasedHsp90 binding to its inhibitor, providing insight into increasingthe efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.PP5 is autoinhibited by intramolecular interactions that can be activated by anumber of cellular factors, includingHsp90. Casein kinase-1δ (CK1δ)-mediated phosphorylation of T362-PP5, was identified as an integral step for PP5 activation, independent of binding to Hsp90. Additionally, the tumor suppressor von Hippel-Lindau (VHL), the substrate recognition component of the VCB-E3-ubiquitin ligase, was found to interact with and multi-monoubiquitinate K185/K199-PP5 for proteasomal degradationin an oxygen-independent manner. Furthermore, VHL-deficient clear cell renal cell carcinoma (ccRCC) cell lines or patient tumors exhibit elevated PP5 levels. Down-regulation of PP5 caused apoptosis inccRCC cells, suggesting a prosurvival role for PP5 in ccRCC.Thisevidence suggests that inhibition of the enzymes that target and catalyze the PTM of Hsp90 and co-chaperones can act synergistically with Hsp90 inhibitors, providingnovel therapeutic strategiesto enhance the efficacy of Hsp90 inhibitors in cancer cells.
    • POSTNATAL ALCOHOL EXPOSURE INFLUENCES ADOLESCENT OLFACTORY RESPONSES TO THE DRUG

      Youngentob, Steven; Harrison, Danielle (2016)
      Human studies illustrate that alcohol exposure while breastfeeding produces a memory of the alcohol scent and modifies behavioral responses to the odor of the drug. The memory and modified behavioral response to alcohol odor suggest that the addictive attributes of alcohol may contribute to patterns of use that increases the risk for alcohol abuse later in life. There is a growing body of evidence that demonstrates prenatal alcohol exposure produces a memory and modified behavioral response to alcohol odor that persists into adolescence, and contributes to alcohol abuse. Given that both postnatal and prenatal alcohol exposure has lasting effects on infants, this study investigated whether rats exposed to alcohol while breastfeeding have a prolonged memory and modified behavioral response to alcohol odor. Long-Evans Hooded rat pups were exposed to alcohol during breastfeeding via the dams' liquid diet. Control animals received ad lib access to an isocaloric, iso-nutritive liquid diet after delivery of their litter up to weaning. To control for effects of malnutrition pair-fed animals were given a control liquid diet equivalent in quantity to the amount their matched animal provided with an alcohol diet consumed the day before. When litters reached adolescence, the behavioral and neurophysiological responses to alcohol odor in a male and female animal from each litter was examined. Relative to controls, animals exposed to alcohol postnatally displayed an altered breathing pattern response to alcohol odor specifically, and an altered breathing pattern and neurophysiological response to novel odorants. The findings of this study builds on the growing body of research that shows the consequences of postnatal alcohol exposure.
    • Preclinical Development of Anti-Cancer Drugs from Natural Products.

      Huang, Ying; Sun, Qing (2014)
      Cancer has been and will continue to be the common concern in the United States and worldwide. As a conventional treatment to fight cancer, new anti-cancer drugs with more efficiency and less toxicity are extremely required. In this study, we have identified two novel compounds with anti-cancer properties from two traditional Chinese medicinal plants. One is Lappaol F that was extracted from the seeds of the plant Actium Lapp L., which has been used in China for centuries as anti-viral and anti-bacterial medicine. Another is M-9 that was extracted from the stem of Marsdenia tenacissima,a plant that has been applied to treat inflammation and cancer in China. Our results showed that Lappaol F inhibited cancer cell growth by regulating a series of cell cycle related proteins and inducing cell cycle arrest at G1 and/or G2 phase. p21 played a critical role in Lappaol F-induced cyclin B1 and cyclin-dependent kinase 1 (CDK1) suppression as well as G2arrest. Lappaol F also induced cell death in a number of cancer cells through caspases activation. Lappaol F-mediated cell growth inhibition was p53-independent. Notably, results from animal studies showed that Lappaol F effectively inhibited tumor growth in vivo, while being well tolerated by the mice. Thus, Lappaol F has a strong potential to be developed as a novel anti-cancer chemotherapeutic. Our studies showed that M-9 successfully sensitized several tumor cells but not non-tumorigenic cells to paclitaxel (Taxol) treatment. Additionally, M-9 reversed chemotherapeutic resistance in a number of multidrug resistant cells. Further results suggested that M-9 functioned, at least to a certain extent, via inhibiting drug efflux by competitively binding to P-glycoprotein (P-gp), a protein that accounts for multidrug resistance. Importantly, results from the in vivostudies demonstrated that M-9 strongly enhanced Taxol-induced growth suppression against xenografts derived from HeLa cells. Moreover, mice tolerated the treatment of Taxol and M-9 well. Therefore, M-9 is a novel chemosensitizer candidate to overcome P-gp-mediated multidrug resistance. Taken together, our studies provide a solid basis for further development of these two compounds as anti-cancer remedies.
    • Psychoactive substance use disorders in adults with attention deficit hyperactivity disorder (ADHD): effects of ADHD and psychiatric comorbidity

      Biederman, J; Wilens, T; Mick, E; Milberger, S; Spencer, T J; Faraone, Stephen V. (American Psychiatric Association Publishing, 1995-11)
      Objective: The authors evaluated the association between attention deficit hyperactivity disorder (ADHD) and psychoactive substance use disorders in adults with ADHD, attending to comorbidity with mood, anxiety, and antisocial disorders. It was hypothesized that psychiatric comorbidity would be a risk factor for psychoactive substance use disorders. Method: Findings for 120 referred adults with a clinical diagnosis ofchildhood-onset ADHD were compared with those for non-ADHD adult comparison subjects (N=268). All childhood and adult diagnoses were obtained by structured psychiatric interviews for DSM-III-R. Rı ıiiIt.ıi There was a significantly higher lifetime risk for psychoactive substance use disorders in the ADHD adults than in the comparison subjects (52% versus 27%). Although the two groups did not differ in the rate ofalcohol use disorders, the ADHD adults had significantly higher rates ofdrug and drug plus alcohol use disorders than the comparison subjects. ADHD significantly increased the risk for substance use disorders independently ofpsychiatric comorbidity. Antisocial disorders significantly increased the risk for substance use disorders independently ofADHD status. Mood and anxiety disorders increased the risk for substance use disorders in both the ADHD and comparison subjects, but more demonstrably in the comparison subjects. Conclusions: Although psychiatric comorbidity increased the risk for psychoactive substance use disorders in adults with ADHD, by itself ADHD was a significant risk factor for substance use disorders. More information is needed to further delineate risk and protective factors mediating the development of substance use disorders in persons with ADHD.
    • 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.
    • RASD2, MYH9, and CACNG2 Genes at Chromosome 22q12 Associated with the Subgroup of Schizophrenia with Non-Deficit in Sustained Attention and Executive Function

      Liu, Yu-Li; Fann, Cathy Shen-Jang; Liu, Chih-Min; Chen, Wei J.; Wu, Jer-Yuarn; Hung, Shuen-Iu; Chen, Chun-Houh; Jou, Yuh-Shan; Liu, Shi-Kai; Hwang, Tzung-Jeng; et al. (Elsevier BV, 2008-11)
      Background: In a previous linkage study of schizophrenia that included Taiwanese samples, the marker D22S278 (22q12.3) was significantly linked to schizophrenia (p .001). Methods: We conducted fine mapping of the implicated genomic region, with 47 validated single nucleotide polymorphism (SNP) markers around 1 Mb of D22S278, in a Taiwanese sample of 218 pedigrees with at least 2 siblings affected with schizophrenia. We examined the association of these SNPs and their haplotypes with schizophrenia and with subgroups defined by the presence and absence of deficits in sustained attention as assessed by undegraded and degraded continuous performance tests (CPTs). We also examined subgroups defined by deficits in categories achieved in the Wisconsin Card Sort Test (WCST). Results: Three of five candidate vulnerability genes (RASD2, APOL5, MYH9, EIF3S7, and CACNG2), which had marginally significant associations with schizophrenia, had significant associations with schizophrenic patients who did not have deficits in sustained attention on the undegraded CPT (RASD2 gene SNP rs736212; p .0008 with single locus analysis) and the degraded CPT (MYH9 gene haplotype 1-1-1-1 of SNP rs3752463 - rs1557540 - rs713839 - rs739097; p .0059 with haplotype analysis). We also found a significant association for patients who showed no deficits in executive function as measured by categories achieved in the WCST (CACNG2 gene haplotype 2-1-1-1 of SNP rs2267360 - rs140526 - rs1883987 - rs916269; p .0163 with haplotype analysis). Conclusions: The genes RASD2, MYH9, and CACNG2 might be vulnerability genes for neuropsychologically defined subgroups of schizophrenic patients.
    • 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.
    • 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.
    • 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.
    • 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.
    • 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.
    • REGULATION OF CELLULAR BIOENERGETICS IN CNS DEMYELINATING DISEASE

      Massa, Paul T.; Minchenberg, Scott (2017)
      Multiple Sclerosis (MS) is a debilitating neurological disease characterized by sclerotic inflammatory demyelination of the white matter tracts in the central nervous system (CNS). There is no “cure” for MS but rather disease modifying treatments that decrease relapse rates and slow disease progression. Due to the lack of insight into the pathogenesis of MS, animal models have been developed to study demyelination in the CNS. Two widely used models of demyelination are experimental autoimmune encephalomyelitis (EAE), and Theiler’s murine encephalomyelitis virus (TMEV). Our studies focused on TMEV mediated demyelination, which was dependent on the expression of the protein tyrosine phosphate SHP-1. SHP-1 is a major negative regulator of cytokine/growth factor signaling and a global deficiency triggers an acute macrophage mediated demyelination in C3H mice. SHP-1 deficient mice are also highly susceptible to systemic inflammation and dysmyelination. Our overall goal was to identify how SHP-1 is mediating susceptibility to inflammatory demyelination. We first demonstrated that SHP-1 deficient oligodendrocytes had increased reactive oxygen species (ROS) production resulting in downregulation of myelin gene expression and oxidation of myelin, a common finding in MS patients. To determine a source of the ROS we investigated how SHP-1 controls metabolic pathways as ROS production is tightly linked to metabolism. To determine how SHP-1 impacts bioenergetics, oligodendrocyte glycolytic and mitochondrial metabolism were quantified using the Seahorse XFe96 analyzer. We determined that SHP-1 enhances oligodendrocyte metabolism, which correlates with its ability to suppress STAT1 activity in oligodendrocytes. We corroborated these results via activation of STAT1 in oligodendrocytes with the proinflammatory cytokine IFN-γ recapitulating the metabolic defects in SHP-1 deficient oligodendrocytes. Based the role of SHP-1 in oligodendrocyte bioenergetics and the importance of macrophage-derived cytokine production during demyelination; we investigated a role for SHP-1 in macrophage bioenergetics. In macrophages, enhanced glycolysis drives activation and proinflammatory cytokine production. TMEV infection specifically induced glycolysis in GM-CSF-derived macrophages lacking SHP-1. This finding may explain why SHP-1 confers susceptibility to macrophage-mediated demyelination after TMEV infection. Overall we demonstrate a novel role for SHP-1 in controlling oligodendrocyte and macrophage bioenergetics that is highly relevant in expanding our understanding of CNS demyelinating disease.
    • RELN AS A CANDIDATE GENE FOR AUTISM SPECTRUM DISORDER (ASD)

      Howell, Brian; Lammert, Dawn (2017)
      Autism spectrum disorder (ASD) affects approximately 1 in 45 people, and is characterized by deficits in social communication and repetitive behaviors. Sequencing advancements have enabled the identification of numerous candidate genes, but precisely how these genes contribute to ASD remains largely unknown. RELNis consistently implicated as a candidate gene for autism. The encoded secreted glycoprotein, Reelin is important for proper brain developmental and postnatal synapse function. Here we examine the molecular and cellular consequences of the de novo RELNmutation R2290C. This mutation falls in a conserved arginine-amino acid-arginine (RXR) motif that is found within the Reelin subrepeat structure. Several other ASD patient mutations fall with in this consensus and all examined reduce Reelin secretion. Based on this we tested two hypothesis: (1) that the mutations reduce Reelin signaling and (2) that they have a gain-of-function consequence, such as ER stress. Using an engineered cell line with a heterozygous RELNR2290C mutation and the RELN Orleans (Orl) mouse line that produces nearly full length Reelin that is defective for secretion, we found evidence for both increased Dab1 and increased PDIA1 expression. Since, like most genes implicated in ASD RELNlikely acts in a multifactorial manner, we investigated whether second site mutations might contribute to ASD-related behaviors. Towards this end we crossed the heterozygous Orl and Shank3b mice to model two hits that are present in at least one ASD proband. We found that the resulting double heterozygousmice had impaired socialization and altered ultrasonic vocalizations. Furthermore, forebrain and cerebellar lysates showed increased PSD-95, identifying a potentially common mechanism and therapeutic target for ASD. These studies are the first to investigate the biological relevance of RELNcoding mutations in ASD.